Method for preparing zero-mode waveguide arrays with coated walls

ABSTRACT

The application relates to methods of manufacture of improved optical containment structures. The invention relates to arrays of zero-mode waveguide structures comprising nanoscale apertures having non-reflective coatings on their walls. The methods provide for selectively coating the walls of the zero mode waveguides, allowing for selective functionalization of the bases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/877,764, filed Sep. 8, 2010, now U.S. Pat. No. 8,906,670, whichclaims the benefit of U.S. Provisional Patent Application No. 61/241,700filed Sep. 11, 2009, which are incorporated by reference herein in theirentirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

Not Applicable.

BACKGROUND OF THE INVENTION

A wide range of analytical operations can benefit from the ability toanalyze the reaction of individual molecules or relatively small numbersof molecules. A number of approaches have been described for providingthese sparsely populated reaction mixtures. For example, in the field ofnucleic acid sequence determination, a number of researchers haveproposed single molecule or low copy number approaches to obtainingsequence information in conjunction with the template dependentsynthesis of nucleic acids by the action of polymerase enzymes.

The various different approaches to these sequencing technologies offerdifferent methods of monitoring only one or a few synthesis reactions ata time. For example, in some cases, the reaction mixture is apportionedinto droplets that include low levels of reactants. In otherapplications, certain reagents are immobilized onto bead or planarsurfaces such that they may be monitored without interference fromtogether reaction components in solution. In still another approach,optical confinement techniques have been used to ascertain signalinformation only from a relatively small number of reactions, e.g., asingle molecule, within and optically confined area.

For arrays of optical confinements it can be desirable to havecomponents to the confinement structures that enable separation of theoptical and solution dimensions. Confinement structures can include, forexample, zero-mode waveguides consisting of subwavelength aperturesextending through a thin cladding layer. Such apertures can provide theability to observe very small volumes of analyte solution, allowing forreliable optical measurements of single molecules within those volumes.While these optical confinements have significantly advanced the abilityto observe single molecules, there is a continuing need for improvedoptical confinement structures, and for methods and systems for usingsuch structures for applications such as nucleic acid sequencing.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the invention comprises a zero-mode waveguide structurecomprising: a transparent substrate having a top surface; an opaquelayer disposed upon the top surface of the transparent substrate; anarray of apertures extending through the opaque layer to the transparentsubstrate whereby the apertures comprise wells having walls and bases,the bases of the wells comprising portions of the top surface of thetransparent substrate; and a non-reflective layer disposed on the wallsof the wells wherein the thickness of the non-reflective layer isgreater than about 5 nm.

In some embodiments the thickness of the non-reflective layer is greaterthan about 10 nm. In some embodiments the non-reflective layer comprisesan oxide. In some embodiments the opaque layer comprises a metal, andthe non-reflective layer comprises an oxide of such metal. In someembodiments the oxide is formed by oxidation of the opaque layer. Insome embodiments the opaque layer comprises a reflective layer. In someembodiments the non-reflective layer comprises phosphorous. In someembodiments the opaque layer comprises aluminum, and the non-reflectivelayer comprises aluminum oxide. In some embodiments the non-reflectivelayer comprises an organic polymer. In some embodiments thenon-reflective layer comprises a silica-based material. In someembodiments the non-reflective layer comprises a silane polymer.

In some embodiments the apertures comprise cylinders. In someembodiments the apertures comprise conical structures.

An aspect of the invention is a zero-mode waveguide structurecomprising: a transparent substrate having a top surface; a reflectivelayer deposed upon the top surface of the transparent substrate; anarray of apertures extending through the reflective layer to thetransparent substrate whereby the apertures comprise wells having wallsand bases, the bases of the wells comprising portions of the top surfaceof the transparent substrate; and a non-reflective layer disposed on thewalls of the wells wherein the thickness of the non-reflective layer isgreater than about 10% of the largest cross-sectional dimension of thewells.

In some embodiments the wells comprise cylindrical structures, wherebythe largest cross-sectional dimensions comprise the diameters of thecylinders. In some embodiments the zero-mode waveguide is less thanabout 80% of the ZMW volume of the zero-mode waveguide. In someembodiments the solution volume of the zero-mode waveguide is less thanabout 75% of the ZMW volume of the zero-mode waveguide. In someembodiments the solution volume of the zero-mode waveguide is less thanabout 70% of the ZMW volume of the zero-mode waveguide.

In one aspect, the invention comprises a zero-mode waveguide comprisingan aperture having a solution cross-sectional area and a ZMWcross-sectional area, wherein the solution cross-sectional area is lessthan about 80% of the cross-sectional area of the zero-mode waveguide.

In some embodiments the solution cross-sectional area is less than about75% of the cross-sectional area of the zero-mode waveguide. In someembodiments the solution cross-sectional area is less than about 70% ofthe cross-sectional area of the zero-mode waveguide.

In one aspect, the invention comprises a method for forming a zero-modewaveguide structure comprising: providing a substrate having a lowertransparent layer and an upper metal layer, wherein the metal layercomprises an array of apertures disposed through the reflective layer tothe transparent layer, the apertures having side walls, and exposing thesubstrate to oxidizing conditions whereby an oxide layer is formed onthe side walls of the apertures under conditions whereby an oxide havinga thickness of greater than 5 nm is produced.

In some embodiments the oxide is formed by chemical oxidation. In someembodiments the oxide is formed electrochemically. In some embodimentsthe oxide is formed with an oxygen plasma. In some embodiments the metalcomprises aluminum, silver, or titanium.

In one aspect, the invention comprises method for forming a zero-modewaveguide array structure comprising: providing an electrochemicalsystem comprising a working electrode, a counter electrode, andoptionally a reference electrode; providing a substrate having a lowertransparent layer and an upper electrically conductive reflective layer,wherein the electrically conductive reflective layer comprises an arrayof apertures disposed through the reflective layer to the transparentlayer, the apertures having side walls, wherein the electricallyconductive reflective layer comprises the working electrode; andapplying a voltage to the working electrode such that a layer ofnon-reflective material is formed onto the side walls of the aperture.

In some embodiments the layer of non-reflective material comprises anoxide. In some embodiments the working electrode is an anode, and theoxide is formed from the oxidation of the electrically conductivereflective layer. In some embodiments the thickness of the oxide isgreater than about 5 nm.

In some embodiments the oxidation of the electrically conductivereflective layer is carried out in the presence of a phosphorouscontaining compound. In some embodiments the phosphorous containingcompound comprises a polymer. In some embodiments the polymer comprisesphosphonate groups.

In one aspect, the invention comprises a method for analyzing aluminescent species comprising: disposing a luminescent species in anaperture that extends through an upper reflective layer that is disposedon a lower transparent layer, wherein the aperture comprises side walls,and a non-reflective layer on the side walls of the aperture having athickness of greater than 5 nm; and detecting emitted light from theluminescent species wherein the emitted light passes through thetransparent layer.

In some embodiments the luminescent species comprises a fluorescentspecies, the method further comprising illuminating the fluorescentspecies with illumination light. In some embodiments the luminescentspecies is associated with a biomolecule. In some embodiments theluminescent species is covalently attached to an enzyme substrate andwherein the emitted light provides information regarding the interactionof the enzyme substrate with the enzyme.

In some embodiments the aperture comprises a complex of a polymeraseenzyme, a template, and a primer, such complex capable of adding acomplementary nucleotide, and wherein the emitted light providesinformation about the addition of the nucleotide. In some embodimentsthe luminescent species is covalently attached to the enzyme, thenucleotide, the template, or the primer. In some embodiments theluminescent species is covalently attached to the nucleotide.

In one aspect, the invention comprises an apparatus for obtainingnucleic acid sequence information comprising: a zero-mode waveguidearray structure comprising. a transparent substrate having a topsurface, and a reflective layer disposed upon the top surface of thetransparent substrate; an array of apertures extending through thereflective layer to the transparent substrate wherein the aperturescomprise wells having walls and bases, the bases of the wells comprisingportions of the top surface of the transparent layer; and anon-reflective layer disposed on the walls of the wells wherein thethickness of the non-reflective layer is greater than about 5 nm; thezero-mode waveguide structure incorporated into a device configured tohold an analysis solution in contact with the zero-mode waveguidestructure, whereby the wells comprise the analysis solution whichcomprises reagents for carrying out reactions for which nucleic acidsequence information can be derived; including polymerase enzyme,nucleotides, and nucleic acid template, the solution further comprisingfluorescent species; an illumination system that illuminates the wellsthrough the transparent layer; a detection system that detects emittedlight over time from the fluorescent species within the wells, whereinthe emitted light passes through the transparent layer; and a computingsystem that analyzes the emitted light over time in order to obtainsequence information.

In some embodiments the fluorescent species are covalently attached tothe nucleotides, and the emitted light over time indicates interactionsbetween the nucleotides and the polymerase enzyme. In some embodimentsthe nucleotides comprise nucleotide analogs.

In one aspect, the invention comprises a method for producing azero-mode-waveguide array comprising: providing an electrochemical cellhaving a working electrode, a counter electrode, and optionally areference electrode, wherein the working electrode comprises a metallicupper layer of a substrate also having a transparent lower layer,wherein the metallic upper layer comprises an array of aperturesextending through metallic upper layer to the transparent lower layer;contacting the working electrode with a solution comprising aphosphorous containing compound; and passing current through theelectrochemical cell whereby a phosphorous containing material isdeposited onto the metallic upper layer of the substrate.

In some embodiments the zero-mode-waveguide array exhibits improvedcorrosion resistance compared to a zero-mode-waveguide array not treatedby the methods of the invention.

In some embodiments the phosphorous containing compound comprisesphosphate or phosphonate functionality. In some embodiments thephosphorous containing compound comprises a polymer. In some embodimentsthe phosphorous containing compound comprises a polymer havingpoly(acrylate), poly(sulfonate), or both poly(acrylate) andpoly(sulfonate) portions. In some embodiments the phosphorous containingcompound comprises polyvinyl phosphonic acid (PVPA), Albritect CP-30,Albritect CP-10, Albritect CP-90, Aquarite ESL, or Aquarite EC4020.

In one aspect, the invention comprises a method for obtaining an islandof functionality at the bases of an array of ZMWs comprising: a)providing an electrochemical system comprising a working electrode, acounter electrode, and optionally a reference electrode; b) providing asubstrate having a lower transparent layer and an upper cladding layer,wherein the cladding layer comprises an array of apertures disposedthrough the reflective layer to the transparent layer, the apertureshaving side walls, wherein the cladding layer comprises the workingelectrode; c) applying a voltage to the working electrode such that alayer of oxide is formed onto the side walls of the aperture; d)attaching functionalizing agent to exposed regions of the transparentlayer within the apertures; and e) dissolving the oxide layer from thewalls of the aperture whereby islands of functionalizing agent areformed within the apertures.

In some embodiments the method further comprising step f) of attaching asingle molecule of interest to the functionalizing agent on thetransparent layer. In some embodiments, step f) is performed after stepe). In some embodiments step f) is performed after step d) and beforestep e).

In some embodiments the single molecule of interest comprises an enzymeor a nucleic acid.

In some embodiments the percentage of aperture having only one singlemolecule of interest is greater than 37%.

In some embodiments the method further comprises performing steps (a),(b), and (c) again after step (e) whereby a second oxide layer is formedto produce an array of apertures having islands of functionalizing agentand oxide layers on the walls.

In some embodiments step (e) of dissolving the oxide layer is carriedout so as to dissolve some of the oxide layer and leave some of theoxide layer undissolved to produce an array of apertures having islandsof functionalizing agent and oxide layers on the walls.

In one aspect, the invention comprises a method for producing an arrayof nanostructures comprising: a) providing a substrate having a topsurface, the top surface having an aperture layer, the aperture layerhaving a plurality of apertures extending through the aperture layer tothe substrate, each of the apertures having one or more cross-sectionaldimension; b) oxidizing the substrate whereby an oxide layer is formedon the aperture layer, whereby a cross sectional dimension of theapertures is brought to 50 nm or smaller; c) treating the substrate witha functionalizing agent whereby the functionalizing agent becomesattached to the exposed portions of the substrate; d) exposing thesubstrate to nanostructures to attach the nanostructures to thefunctionalizing agent attached to the substrate; and e) dissolving theoxide layer.

In some embodiments the nanostructures comprise nanoparticles. In someembodiments, step (d) is performed after step (c) and before step (e).In some embodiments, step (d) is performed after step (e). In someembodiments, in step (b) the cross-sectional dimension is brought to 10nm or smaller. In some embodiments the aperture layer comprises a metal.

In one aspect, the invention comprises a method for forming an array ofnanopores comprising: a) providing a substrate comprising an array ofapertures extending therethrough, each of the apertures having one ormore cross-sectional dimension; and b) oxidizing the substrate wherebyan oxide layer is formed on the substrate and whereby the formed oxidelowers one or more cross sectional dimensions of the apertures to 20 nmor less.

In some embodiments step (b) the formed oxide lowers the aperturedimensions to 5 nm or less. In some embodiments the substrate comprisesa metal. In some embodiments the substrate comprises silicon.

In one aspect the invention provides method for producing an island offunctionalizing agent in an array of ZMW's comprising: a) providing asubstrate having on its surface a cladding layer, wherein the claddinglayer comprises an array of apertures disposed through the claddinglayer to the substrate, the apertures having side walls; b) selectivelygrowing an aperture constriction layer on the cladding layer such thatthe aperture constriction layer extends in from the side walls of theaperture to reduce the cross-sectional dimensions of the aperture. c)attaching functionalizing agent to exposed regions of the substratewithin the apertures; and d) removing the aperture constriction layerwhereby an array of apertures, each having an island of functionalizingagent is produced.

In some embodiments the aperture constriction layer comprises a polymer.In some embodiments the aperture constriction layer comprises a metaloxide.

In some embodiments the aperture constriction layer comprises a metal.In some embodiments the step of selectively growing an apertureconstriction layer comprises growing a polymer from the cladding with apolymerization reaction extending from the surface of the cladding. Insome embodiments step of selectively growing an aperture constrictionlayer comprises growing an oxide onto the cladding by connecting thecladding to a voltage source under conditions such that controlledoxidation of the cladding occurs. In some embodiments the step ofselectively growing an aperture constriction layer compriseselectrodepositing a material onto the cladding by connecting thecladding to a voltage source and providing the current required forelectrodeposition.

In some embodiments the substrate comprises a transparent material. Insome embodiments the transparent material comprises quartz or fusedsilica. In some embodiments the substrate comprises silicon. In someembodiments the cladding comprises a metal. In some embodiments themetal comprises aluminum.

In some embodiments the apertures have a cylindrical profile and have adiameter between about 70 nm and 300 nm. In some embodiments the islandshave diameter between about 5 nm and about 50 nm.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an illustration of a zero-mode-waveguide.

FIG. 2 provides an illustration single molecule nucleic acid sequencingin an optical confinement.

FIG. 3 shows a ZMW with (B) and without (A) a non-reflective layer onits walls.

FIG. 4 illustrates optical illumination and detection of a fluorescentspecies in a ZMW with (B) and without (A) a non-reflective layer on itswalls.

FIG. 5 shows an optical model of illumination intensity at the base of aZMW without a reflective layer on its walls.

FIG. 6 shows an optical model of illumination intensity at the base of aZMW having a reflective layer on its walls.

FIG. 7 shows cross-sections of some exemplary ZMWs of the inventionhaving non-reflective layers on their walls.

FIG. 8 shows an apparatus or system of the invention.

FIG. 9 (A)-(C) illustrates the production of an oxide layer on a ZMWstructure.

FIG. 9 (D)-(E) illustrates the production of oxide on a ZMW structure inwhich the aperture extends into the transparent substrate.

FIG. 10 illustrates a process for producing the non-reflective layer ofthe invention by depositing a conformal coating.

FIG. 11 illustrates a process for producing the non-reflective layer ofthe invention for a ZMW having an aperture that extends into thetransparent substrate by depositing a conformal coating.

FIG. 12 illustrates a process for producing a non-reflective layer ofthe invention using a planar coating.

FIG. 13 illustrates an embodiment of a method of the invention forproducing an island of functionality within the ZMW by growing anaperture constriction layer.

FIG. 14 illustrates an embodiment of a method of the invention forproducing an island of functionality within the ZMW using oxidation ofthe cladding.

FIG. 15 illustrates an embodiment of a method of the invention forproducing nanopores.

FIG. 16 is a plot of current density versus voltage for theelectrochemical anodization of a zero-mode-waveguide.

FIG. 17 is a plot of impedance spectroscopy data showing improvedstability for an electrochemically anodized zero-mode-waveguide.

FIGS. 18 and 19 show a TEM images of a cross section of a ZMW structureof the invention prepared in the presence of PVPA by applying 10V.

FIG. 20 shows a TEM image of cross-section of a ZMW structure producedby oxidation in the presence of PVPA at 15 V.

FIG. 21 shows a TEM image of cross-section of a ZMW structure producedby oxidation in the presence of PVPA at 25 V.

FIG. 22 shows a plot of ZMW illumination intensity versus the refractiveindex of the wall material for a green laser and a red laser.

FIG. 23 shows single molecule sequencing data for sequencing runscarried out on ZMW arrays anodized at 10V and 20V, and on a control ZMWarray not anodized.

FIG. 24 shows the signal to noise measured for sequencing runs carriedout on ZMW arrays anodized at 10V and 20V, and on a control ZMW arraynot anodized.

FIG. 25 shows the distribution of signal to noise for sequencing runscarried out on ZMW arrays anodized at 10V and 20V, and on a control ZMWarray not anodized.

DETAILED DESCRIPTION OF THE INVENTION

General

In some aspects, the present invention provides optical confinementstructures such as zero-mode waveguide (ZMW) structures capable ofimproved performance. The present invention also provides for arrays ofsuch ZMWs, methods for making and using, and systems incorporating suchimproved ZMWs. The ZMW structures of the present invention have wallscomprising non-reflective material, and in particular, walls comprisinga non-reflective material having a higher refractive index than that ofthe medium within of the ZMW during optical analysis. ZMW structures canbe used to analyze solutions containing luminescent species disposedinside or in close proximity to the ZMW. In a ZMW without anon-reflective layer, the solution volume and the ZMW volume, whichcontrols the optical performance, are substantially the same. The use ofa ZMW having a layer of non-reflective material on its walls allows fordecoupling the volume containing the solution containing the luminescentspecies from the volume of the ZMW structure that controls thepropagation of light. The layer of non-reflective material on the wallscan also act to hold or constrain the luminescent species within aregion within the center of the ZMW where it can effectively beilluminated and/or detected. In addition, where the walls have a higherrefractive index than the medium within the ZMW, the illumination lightintensity can be more effectively directed into the portion of the ZMWin which the luminescent species resides. The present invention alsoprovides methods for obtaining islands of functionality within nanoscaleapertures and the use of such islands for binding single molecules orsingle particles within the apertures.

The basic functional structure of a ZMW is schematically illustrated inFIG. 1. As shown, a ZMW structure 100 is provided that includes acladding layer 102 deposited upon a transparent layer 104. An apertureor core 106 is disposed through the cladding layer to expose thetransparent layer 104 below. The aperture 106 has a base 120 thatcomprises the top surface of the transparent layer 104. As shown in FIG.1, the base 120 of the aperture 106 is at the same level as the planarsurface of the transparent layer 104. In some cases, the base 120 of theaperture 106 is not at the same level, and can be above or below theplanar surface of the transparent layer 104 outside of the aperture. Forexample, in some cases, the base of the aperture can be below the levelof the surface of the transparent 104, extending into the transparentlayer 104. The core is dimensioned to provide optical confinement byattenuating or preventing propagation of electromagnetic radiation thatfalls below a cut-off frequency through the core. Instead, the lightonly penetrates a short distance into the core, illuminating arelatively small volume, indicated as bounded by the dashed line 108. Byproviding reactants of interest within the observation volume, e.g.,enzyme 110 and substrate 112, one can selectively observe theiroperation without interference from reactants, e.g., substrates 114outside the observation volume, e.g., above line 108. It will beunderstood by those in the art the intensity will fall off in the corewith a certain function, e.g. exponentially, and that line 108 does notnecessarily represent a line above which no light penetrates, but canrepresent, for example, a line at which the light falls to a certainabsolute or relative intensity level.

ZMW structures can be used in order to observe very small quantities ofanalytes, and have been shown to provide information on the presence andbehavior of an analyte to the level of a single molecule. The ability toobserve a single molecule in real time allows for carrying out singlemolecule sequencing, for example single molecule nucleic acidsequencing.

In the context of single molecule nucleic acid sequencing analyses, asingle immobilized nucleic acid synthesis complex, comprising apolymerase enzyme, a template nucleic acid, whose sequence one isattempting to elucidate, and a primer sequence that is complementary toa portion of the template sequence, is observed to identify individualnucleotides as they are incorporated into the extended primer sequence.Incorporation is typically monitored by observing an opticallydetectable label on the nucleotide, prior to, during or following itsincorporation. In some cases, such single molecule analyses employ a“one base at a time approach”, whereby a single type of labelednucleotide is introduced to and contacted with the complex at a time.Upon incorporation, unincorporated nucleotides are washed away from thecomplex, and the labeled incorporated nucleotides are detected as a partof the immobilized complex.

In some instances, only a single type of nucleotide is added to detectincorporation. These methods then require a cycling through of thevarious different types of nucleotides (e.g., A, T, G and C) to be ableto determine the sequence of the template. Because only a single typenucleotide is contacted with the complex at any given time, anyincorporation event is by definition, an incorporation of the contactednucleotide. These methods, while somewhat effective, generally sufferfrom difficulties when the template sequence includes multiple repeatednucleotides, as multiple bases may be incorporated that areindistinguishable from a single incorporation event. In some cases,proposed solutions to this issue include adjusting the concentrations ofnucleotides present to ensure that single incorporation events arekinetically favored.

In other cases, multiple types of nucleotides are added simultaneously,but are distinguishable by the presence on each type of nucleotide of adifferent optical label. Accordingly, such methods can use a single stepto identify a given base in the sequence. In particular, all fournucleotides, each bearing a distinguishable label, is added to theimmobilized complex. The complex is then interrogated to identify whichtype of base was incorporated, and as such, the next base in thetemplate sequence.

In some cases, these methods only monitor the addition of one base at atime, and as such, they (and in some cases, the single nucleotidecontact methods) require additional controls to avoid multiple basesbeing added in any given step, and thus being missed by the detectionsystem. Typically, such methods employ terminator groups on thenucleotide that prevent further extension of the primer once onenucleotide has been incorporated. These terminator groups are typicallyremovable, allowing the controlled re-extension after a detectedincorporation event. Likewise, in order to avoid confounding labels frompreviously incorporated nucleotides, the labeling groups on thesenucleotides are typically configured to be removable or otherwiseinactivatable.

In another process, single molecule primer extension reactions aremonitored in real-time, to identify the continued incorporation ofnucleotides in the extension product to elucidate the underlyingtemplate sequence. In such single molecule real time (or SMRT™)sequencing, the process of incorporation of nucleotides in a polymerasemediated template dependent primer extension reaction is monitored as itoccurs. In preferred aspects, the template/polymerase primer complex isprovided, typically immobilized, within an optically confined region,such as a zero mode waveguide, or proximal to the surface of atransparent substrate, optical waveguide, or the like (see e.g., U.S.Pat. Nos. 6,917,726, and 7,170,050 and Published U.S. Patent ApplicationNo. 2007-0134128, the full disclosures of which are hereby incorporatedherein by reference in their entirety for all purposes). The opticallyconfined region is illuminated with an appropriate excitation radiationfor the fluorescently labeled nucleotides that are to be used. Becausethe complex is within an optically confined region, or very smallillumination volume, only the reaction volume immediately surroundingthe complex is subjected to the excitation radiation. Accordingly, thosefluorescently labeled nucleotides that are interacting with the complex,e.g., during an incorporation event, are present within the illuminationvolume for a sufficient time to identify them as having beenincorporated. A schematic illustration of this sequencing process isshown in FIG. 2. As shown in FIG. 2A, an immobilized complex 202 of apolymerase enzyme, a template nucleic acid and a primer sequence areprovided within an observation volume (as shown by dashed line 204) ofan optical confinement, of e.g., a zero mode waveguide 206. As anappropriate nucleotide analog, e.g., nucleotide 208, is incorporatedinto the nascent nucleic acid strand, it is illuminated for an extendedperiod of time corresponding to the retention time of the labelednucleotide analog within the observation volume during incorporationwhich produces a signal associated with that retention, e.g., signalpulse 212 as shown by the A trace in FIG. 1B. Once incorporated, thelabel that attached to the polyphosphate component of the labelednucleotide analog, is released. When the next appropriate nucleotideanalog, e.g., nucleotide 210, is contacted with the complex, it too isincorporated, giving rise to a corresponding signal 214 in the T traceof FIG. 213. By monitoring the incorporation of bases into the nascentstrand, as dictated by the underlying complementarity of the templatesequence, one can obtain long stretches of sequence information of thetemplate. Further, in order to obtain the volumes of sequenceinformation that may be desired for the widespread application ofgenetic sequencing, e.g., in research and diagnostics, higher throughputsystems are desired.

By way of example, in order to enhance the sequencing throughput of thesystem, multiple complexes are typically monitored, where each complexis sequencing a separate template sequence. In the case of genomicsequencing or sequencing of other large DNA components, these templateswill typically comprise overlapping fragments of the genomic DNA. Bysequencing each fragment, one can then assemble a contiguous sequencefrom the overlapping sequence data from the fragments. In preferredaspects, the various different complexes are provided arrayed upon asubstrate. Such arrayed complexes may be provided within optically orstructurally confined structures, e.g., zero mode waveguides, or theymay be patterned on a surface. Alternatively, they may be randomlydisposed over a surface but subjected to targeted arrayed illumination,or detection, such that only complexes within an array pattern on thesurface are monitored. For purposes of discussion herein, bothconfigurations are referred to herein as the monitoring of arrayedcomplexes, or the like.

FIG. 3 illustrates how having non-reflective layers on the walls of theZMW decouples the solution volume from the ZMW volume. As used herein,the term ZMW volume refers to the volume within the aperture or corewhich extends through the opaque cladding layer disregarding thenon-reflective walls. The solution volume, as used in this context, isthe volume that the solution would take up in the ZMW, that is, thevolume within the ZMW inside the non-reflective walls. We note that insome cases a ZMW of the invention can be used without a solution present(e.g. detecting a gaseous luminescent species). Thus, the solutionvolume as used herein may or may not contain a solution. FIGS. 3 (A) and3(B) represent cross-sections through ZMWs having a cladding layer 302disposed on a transparent substrate 301. The ZMW in Figure (A) (ZMW(A))has substantially no non-reflective layer on its walls, thus thecross-sectional dimension of the solution volume (d_(Soln)) issubstantially the same as the cross sectional dimension of the ZMW(d_(ZMW)). Therefore, the cross-sectional area of the solution volumewill be substantially the same as the cross-sectional area of the ZMWvolume, and the solution volume will be substantially the same as theZMW volume.

In the ZMW of Figure (B) (ZMW(B), representing a ZMW of the invention,the walls of the ZMW have a non-reflective material disposed upon them.As shown here, the cross-sectional dimension (d_(ZMW)) is the same forZMW(A) and ZMW(B) and thus the cross sectional areas and volumes arealso the same. However, by incorporating a non-reflective layer 303 intothe inner walls of ZMW(B), the solution cross-sectional dimension(d_(Soln)) of ZMW(B) is smaller than the corresponding cross-sectionaldimension of ZMW(A), and the solution cross-sectional area and solutionvolume are smaller in ZMW(B) the corresponding cross-sectional area andsolution volume of ZMW(A). The dotted line 330 represents theilluminated region within the ZMW. FIG. 3 illustrates that theillumination region within the ZMW is not substantially changed by theaddition of the non-reflective walls. The illumination region willremain unchanged where the refractive index of the material on the wallsis the same as the refractive index of the medium within the solutionvolume. As described in more detail below, where the refractive index ofthe walls is larger than the refractive index of the medium within thesolution volume, the illumination region can change to direct a higherportion of the illumination energy into the region of the solutionvolume within the ZMW in ways which can be beneficial. We have foundthat the ability to decouple the solution volume from the ZMW volume inthis manner has a number of useful and non-obvious benefits.

One advantage of the ZMW of the present invention is a reduction in thelevel of background light, for example background fluorescence, from thesolution. This benefit can be seen by referring to FIG. 4, in which,similar to FIG. 3, ZMW(B) has a non-reflective layer on the walls, andthereby has a smaller solution cross-sectional area and a smallersolution volume than ZMW(A), but ZMW(A) and ZMW(B) have the samegeometry with respect to the opaque layer: i.e. ZMW(A) and ZMW(B) eachhave the same ZMW cross-sectional area and the same ZMW volume. FIG. 4represents a ZMW used within a system in which fluorescence is used toanalyze molecules within the ZMW. The illumination (or excitation) light410 is introduced from below, through the transparent substrate 401 intothe ZMW. Fluorescent species within the ZMW that interact with theillumination light may emit fluorescent emitted light 420, which can bedetected from below the transparent layer with a detector. In FIG. 4,the ZMW's have bound to the transparent substrate 401 a single molecule404 such as a single nucleic acid polymerase molecule used for singlemolecule sequencing as described herein. In addition, the ZMW's have insolution background fluorescent species 406 which are capable ofemitting fluorescent light. This background fluorescent light isgenerally undesirable as the background light generally does not provideuseful information to the analysis, and such light observed by thedetector can contribute to the noise in the system. As is illustrated inFIG. 4, since the solution volume in ZMW(B) is smaller than the solutionvolume in ZMW(A), the number of background fluorescent species will besmaller, and therefore the background fluorescent light will be lower inZMW(B), resulting in a higher signal-to-noise ratio (SNR) for detectionof the fluorescent signals of interest, such as fluorescent signalsassociated with molecules bound by a polymerase enzyme within theobservation region.

The comparison of ZMW's with and without a non-reflective layer made inreference to FIGS. 3 and 4 compare structures with the same ZMWdimensions and a smaller solution volume. The benefits of thenon-reflective layer can be further appreciated by considering twostructures, each having the same solution volume but one having anon-reflective layer and one having no such layer. In this case, the ZMWstructure with the non-reflective layer will have a ZMW with a largercross section. The use of a ZMW with a larger cross-section can bebeneficial in increasing the level of emission from a luminescentspecies within the solution volume. Controlling this effect can beparticularly useful, for example, when analyzing a reaction havingmultiple luminescent species, such as a nucleic acid sequencing reactionhaving multiple fluorescently labeled nucleotides. The opticalcharacteristics of a given ZMW can vary with the wavelength of the lightthat is interacting with the ZMW, either the illumination or theemission light. The ZMW structures of the invention thus allow forindependently tuning the waveguide portion and the solution containingportion of the ZMW structure.

Another advantage of the ZMW of the present invention is that thesolution volume is moved into the center of the ZMW and away from thewalls. The placement of the luminescent species of interest toward thecenter of the ZMW and away from the walls can be useful for severalreasons. One reason is that the illumination light can be of higherintensity and higher consistency toward the middle of the ZMW. Anotheris that the collection of emitted light can be more effective for anemitting species when it is away from the walls. It is known, forexample, that a fluorescent species can have its fluorescencesignificantly attenuated, and in some cases completely quenched when itis positioned very near or directly on a reflective surface such as thesurface of a metal. Thus the non-reflective material keeps the emittingspecies away from the walls where its emission can more effectivelydetected.

Having a fluorescent species of interest away from the walls can also beimportant in order to avoid having the fluorescent species in areaswhere there are areas of very high illumination intensity, orillumination “hot spots”. We have found by optical modeling of the ZMWthat illumination hot spots can occur at the edges of the ZMW such asedges at the base of the ZMW where the wall meets the transparentsubstrate. FIG. 5 shows a result of such optical modeling for a ZMWwithout a non-reflective layer on its walls. The ZMW in FIG. 5 has afused silica transparent substrate with a refractive index of about1.46, and an aluminum cladding layer with a thickness of 110 nm. The ZMWis cylindrical aperture having a diameter of 100 nm. The ZMW is filledwith an aqueous medium having a refractive index of about 1.33. The ZMWis illuminated through the transparent substrate with 532 nm lightpolarized in the x-direction. FIG. 5 shows 2-dimensional plot ofintensity versus position along the base of the ZMW. The higher theintensity, the lighter the color, consistent with the scale shown on theright side of the figure. As can be seen in FIG. 5, the optical modelingshows high intensity hot spots at the edges of the ZMW at its base. Afluorescent species in the region of the hot spot would experience veryhigh light intensity. In addition, and for the reasons described above,the fluorescent emission of this fluorescent species can be attenuatedor quenched due to its proximity to the reflective cladding.

FIG. 6 shows the optical modeling results for the same ZMW as in FIG. 5,but having a 20 nm thick layer of a non-reflective material with arefractive index of about 2.5 on the ZMW walls. FIG. 6 shows that thesolution volume region inside of the non-reflective material layer isnot exposed to the hot spots as was the case for the solution volume inFIG. 5. It is well known that photodamage to luminescent species withinthe ZMW can inhibit the performance of the ZMW. Such photodamage can,for example, inhibit or destroy the activity of a polymerase enzyme usedfor nucleic acid sequencing. Thus, eliminating hot-spots, andcontrolling the illumination intensity within the ZMW can providesignificant improvements in the quality of sequencing data obtained fromreactions carried out within the ZMW.

FIG. 6 also illustrates an advantage of having non-reflective walls witha higher refractive index than that of the medium within the solutionvolume. It can be seen in FIG. 6 that the intensity of the light withinthe solution region is generally higher than the intensity in theregions of the non-reflective layers. The relative intensity of light inthe non-reflective layer and the light in the solution region can becontrolled by controlling the relative refractive indices of the mediumin the solution volume and the non-reflective layers. Generally, thehigher the ratio of wall refractive index to solution refractive index,the more light intensity which will be directed into the solutionvolume. FIG. 6 shows that the illumination light intensity isconcentrated into the region inside the non-reflective walls of higherrefractive index at the base of the ZMW. Optical modeling shows thatthere can be a benefit of increased illumination intensity also forstructures where the aperture within the ZMW extends into thetransparent substrate, for example for the structures such as that shownin FIG. 7(E).

ZMW with Non-Reflective Layers on the Walls

Zero mode waveguides (ZMWs) are generally characterized by the existenceof a core surrounded by a cladding, where the core is dimensioned suchthat it precludes a substantial amount of electromagnetic radiation thatis above a cut-off frequency from propagating through the core. As aresult, when illuminated with light of a frequency below the cutofffrequency, the light will only penetrate a short distance into the core,effectively illuminating only a small fraction of the core's volume. Inaccordance with the present invention, the ZMW core comprises an emptyor preferably fluid filled cavity surrounded by a non-reflective layerand then the cladding layer. This core then provides a zone or volume inwhich a chemical, biochemical, and/or biological reaction may take placethat is characterized by having an extremely small volume, which can beused to effectively observe a single molecule or set of reacting singlemolecules. ZMWs, their fabrication, structure, and use in analyticaloperations are described in detail in U.S. Pat. No. 6,917,726 andLevene, et al., Science 299(5607):609-764 (2003), the full disclosuresof which are hereby incorporated herein by reference in their entiretyfor all purposes.

In the context of chemical or biochemical analyses within ZMWs as wellas other optical confinements, it is clearly desirable to ensure thatthe reactions of interest are taking place within the opticallyinterrogated portions of the confinement, at a minimum, and preferablysuch that only the reactions of a single molecule is occurring within aninterrogated portion of an individual confinement. A number of methodsmay generally be used to provide individual molecules within theobservation volume. A variety of these are described in co-pending U.S.patent application Ser. No. 11/240,662, filed Sep. 30, 2005,incorporated herein by reference in its entirety for all purposes, whichdescribes, inter alia, modified surfaces that are designed to immobilizeindividual molecules to the surface at a desired density, such thatapproximately one, two, three or some other select number of moleculeswould be expected to fall within a given observation volume. Typically,such methods utilize dilution techniques to provide relatively lowdensities of coupling groups on a surface, either through dilution ofsuch groups on the surface or dilution of intermediate or final couplinggroups that interact with the molecules of interest, or combinations ofthese.

One aspect of the invention is a zero-mode waveguide structurecomprising: a transparent substrate having a top surface; an opaquelayer disposed upon the top surface of the transparent substrate; anarray of apertures extending through the opaque layer to the transparentsubstrate wherein the apertures comprise wells having walls and bases,the bases of the wells comprising portions of the top surface of thetransparent substrate; and a non-reflective layer disposed on the wallsof the wells wherein the thickness of the non-reflective layer isgreater than about 5 nm. In some cases the non-reflective layer isdisposed on the walls of the wells wherein the thickness of thenon-reflective layer is greater than about 10% of the largestcross-sectional dimension of the wells.

Typically, the ZMW aperture has at least one cross-sectional dimension,e.g., diameter, which is sufficiently small that light entering thewaveguide is prevented in some measure from propagating through thecore, effectively resulting in a very small portion of the core and itscontents being illuminated, and/or emitting optical signals that exitthe solution volume of the core. In the case of optical signals (andexcitation radiation), the ZMW cores will typically be between about 1nm and about 300 nm, between about 10 and about 200 nm, or between about50 and about 150 nm in diameter where light in the visible range isused.

The individual confinement in the array can provide an effectiveobservation volume less than about 1000 zeptoliters, less than about900, less than about 200, less than about 80, less than about 10zeptoliters. Where desired, an effective observation volume of less than1 zeptoliter can be provided. In a preferred aspect, the individualconfinement yields an effective observation volume that permitsresolution of individual molecules, such as enzymes, present at or neara physiologically relevant concentration. The physiologically relevantconcentrations for many biochemical reactions range from micro-molar tomillimolar because most of the enzymes have their Michaelis constants inthese ranges. Accordingly, a preferred array of optical confinements hasan effective observation volume for detecting individual moleculespresent at a concentration higher than about 1 micromolar (μM), or morepreferably higher than 50 μM, or even higher than 100 μM.

A zero-mode-waveguide can provide an optical guide in which the majorityof incident radiation is attenuated, preferably more than 80%, morepreferably more than 90%, even more preferably more than 99% of theincident radiation is attenuated. As such high level of attenuation, nosignificant propagating modes of electromagnetic radiation exist in theguide. Consequently, the rapid decay of incident electromagneticradiation at the entrance of such guide provides an extremely smallobservation volume effective to detect single-molecules, even when theyare present at a concentration as high as in the micromolar range.

The ZMW internal cavity (i.e., the core) surrounded by the cladding mayadopt a convenient size, shape or volume so long as propagating modes ofelectromagnetic radiation in the guide is effectively prevented. Thecore typically has a lateral dimension less than the cutoff wavelength(λ_(c)). For a circular guide of diameter d and having a clad of perfectconductor, λ_(c) is approximately 1.7 times d. The cross sectional areaof the core may be circular, elliptical, oval, conical, rectangular,triangular, polyhedral, or in any other shape. Although uniform crosssectional area is generally preferred, the cross sectional area may varyat any given depth of the guide if desired. For example, in some casesthe ZMW has a conical shape. The shape of the ZMW core, and the shape ofthe solution volume are generally similar, but in some cases they can bedifferent. For example, in some embodiments, the ZMW core iscylindrical, and the shape of the solution volume defined bynon-reflective walls is conical.

In some embodiments, the ZMW core is non-cylindrical. In one aspect ofthis embodiment, a non-cylindrical core comprises an opening on theupper surface and a base at the bottom surface, wherein the opening isnarrower in lateral dimension than the base. In another aspect, theopening at the base is narrower than the opening at the surface. Thisconfiguration significantly restricts the diffusion of reactants, andhence increases the average residence time in the observation volume.Such configuration can be useful, for example, for measuring theassociation rate constant (on-rate) of a chemical reaction. In anotheraspect, the core comprises an opening that is wider in lateral dimensionthan the base. Such configuration allows easier access to largemolecules that impose a steric or entropic hindrance to entering thestructure if the open end of the zero mode waveguide was as small as thebase needed to be for optical performance reasons. Examples include theaccessibility for long strand polyelectrolytes such as DNA moleculesthat are subject to entropic forces opposing entry into small openings.

FIG. 7 shows cross-sections of some specific embodiments of ZMWstructures having non-reflective layers on their walls. The ZMW shown inFIG. 7(A) has a layer of non-reflective material on the walls of the ZMWaperture and also on the top of the cladding layer. In some cases, thecross sections represent an aperture with a circular profile. Thecross-sections can also represent profiles with other shapes including aslit, ellipse, rectangle, star, or any other suitable shape. This typeof structure can result, for example, where the non-reflective layercomprises an oxide that is specifically grown onto a metal claddinglayer, e.g. by thermal or electrochemical oxidation. The structure canalso be produced by selective deposition onto the cladding layer or byfirst conformally coating the non-reflective material and second,removing the portion of the non-reflective material on the transparentsubstrate. In the ZMW shown in FIG. 7(B), the non-reflective layercovers the walls of the ZMW, the top of the cladding, and the top of thetransparent substrate within the ZMW. This type of structure can becreated, for example, by conformally coating a ZMW structure with anon-reflective material. In some cases, as shown in FIG. 7(C), the ZMWaperture will extend into the transparent substrate, and thenon-reflective layer will be specifically on the opaque cladding layer,in some cases extending over the a portion of the aperture that extendsinto the transparent substrate. FIG. 7 (D) shows a ZMW in which thenon-reflective layer is only on the inside walls of the ZMW, and not onthe top of the surface of the cladding. In FIG. 7(E) the portion of theaperture that can hold the solution extends into the transparentsubstrate and the non-reflective layer does not extend over the portionthat extends into the transparent substrate.

FIG. 7(F) shows a ZMW in which the ZMW aperture extends into thetransparent substrate and the non-reflective layer extends into theportion of the aperture that extends into the transparent substrate. InFIG. 7(G) the ZMW aperture has straight side walls (which for a ZMW witha spherical cross section would constitute a cylinder), and thenon-reflective layer is applied so as to have angled side-walls (which,for a solution volume with a spherical cross section would constitute aconical structure). FIG. 7(H) is similar to FIG. 7(G), but in which thesolution containing portion extends into the transparent layer. InFigure (I), the ZMW aperture extends into the transparent substrate, andthe non-reflective layer coats the inside walls of the ZMW, the base ofthe ZMW, and the top surface of the cladding layer. In Figure (J), theZMW has angled sidewalls (which for a ZMW with a cylindrical crosssection would constitute a conical structure), and the non-reflectivelayer also has angled sidewalls. FIG. 7(K) shows a ZMW similar to thatin FIG. 7(J), but with the solution containing portion extending intothe transparent layer. The ZMW of FIG. 7(L) is similar to that of FIG.7(D), but having a non-reflective layer which is thicker. FIG. 7(L)illustrates that the methods of the invention can be used to lower thesolution volume significantly and also to position a luminescent speciesat or near the center of the ZMW. It will be appreciated by thoseskilled in the art that the ZMW of the invention can be comprise acombination of two or more of the structures shown in FIG. 7.

The height of the ZMW will generally be the thickness of the claddinglayer, and the ZMW aperture may extend into the transparent layer. Theheight of the ZMW can be between about 5 nm and 500 nm, between 20 nmand 300 nm, or between 50 nm and 150 nm. In some cases, the height ofthe ZMW is between about 80 nm and 140 nm.

The thickness of the non-reflective layer will generally be greater thanabout 5 nm. It is known, for example, that the native oxide layer on thesurface of an aluminum metal can have a thickness of between about 3 to4 microns. The thickness of the non-reflective layer will generally begreater than the thickness of this native oxide coating. It will beunderstood that the best thickness can depend on the diameter of the ZMWthat is used and the use to which the ZMW is put. In some cases, forexample, while a greater thickness of the non-reflective layer may beuseful for improved optical properties, the greater thickness may resultin a solution volume which is too small to accommodate the species to beanalyzed, for example, the enzyme and/or its substrate. The structuresand methods of the invention allow for adjusting the thickness of thenon-reflective layer and the dimensions of the ZMW in order to improvethe overall performance of the system which incorporates the ZMW, forexample the analysis of biomolecules and nucleic acid sequencing.

In some cases, the non-reflective coating has a thickness of betweenabout 5 nm and about 50 nm, between about 8 nm and 40 nm, and betweenabout 10 nm and about 30 nm. In some cases, a ZMW having across-sectional dimension of about 50 nm to about 105 nm has anon-reflective coating of about 10 nm to about 30 nm of thickness. Thethickness of the non-reflective layer is generally maintained such thata solution volume is maintained inside of the ZMW.

The non-reflective layer will generally result in a cross-sectionaldimension within the non-reflective layer being less than thecorresponding cross-sectional dimension of the ZMW. For the ZMW's of theinvention, the solution volume within the non-reflective area of the ZMWwill have a cross-sectional dimension that is about 10% to about 95%,from about 20% to about 80%, or between about 25% to about 50% of thecorresponding cross-sectional dimension of the ZMW. Where the ZMW andthe solution volume within the non-reflective layer in the ZMW eachcomprise cylindrical structures with circular cross sections, forexample, the cross-sectional dimension would be the diameter of thecircular cross-section. In some cases the cross-sectional dimensionswill vary with height, in which case the average or mediancross-sectional dimension can be used. In other cases, thecross-sectional dimension at a given height, such as at the base of theZMW can be used.

The non-reflective coating will take up a portion of the cross-sectionalarea which would be available to a solution if the non-reflective layerwas not present, thus lowering the solution volume within the ZMW. Insome cases, the ZMW of the invention will have a cross-sectional areainside the non-reflective layer (the solution volume) that comprisesfrom about 10% to about 90%, from about 30% to about 80%, or betweenabout 30% to about 50% of the corresponding cross-sectional area of theZMW. The cross-sectional area of a ZMW may vary with height. In somecases, the average or median cross sectional area of the ZMW is used todetermine the relative amount of the non-reflective layer. In somecases, the relative cross sectional areas at a given height, such as atthe base of the ZMW can be used.

The non-reflective layer results in a solution volume within thenon-reflective layer in the ZMW that is smaller than the ZMW volume. Insome cases, the ZMW of the invention will have a volume inside thenon-reflective layer (the solution volume) that comprises between about10% to about 90%, between about 30% to about 80%, or between about 30%to about 50% of the ZMW volume.

The cladding is typically made of materials that prevent any significantpenetration of the electric and the magnetic fields of anelectromagnetic radiation that is opaque and/or reflective materials.Suitable materials for fabricating the cladding include but are notlimited to metals, metal oxides, alloys, conducting materials andsemi-conducting materials, and any combination thereof. The claddinglayer can comprise a metal such as aluminum, copper, gold, silver,chromium, titanium or mixtures thereof.

The transparent substrate can comprise inorganic materials, organicmaterials, or composite materials with both organic and inorganicmaterials. The transparent material is typically a rigid material whichcan keep the reactive regions in fixed positions during observation.Silica based materials, such fused silica are preferred materials, forexample, where semiconductor or MEMS processing methods are used toproduce the micromirror arrays. The transparent substrate may alsocomprise inorganic oxide materials and glasses. The transparentsubstrate material may be a heterogeneous material, such as a materialhaving multiple layers. In some cases, for example, the transparentsubstrate may comprise a dielectric stack. Transparent polymericmaterials can also be used. It is typically desired that the transparentmaterial exhibit low levels of autofluorescence. Suitable transparentpolymers comprise, for example, methacrylate polymers such as PMMA,polycarbonates, cyclic olefin polymers, sytrenic polymers,fluorine-containing polymers, polyesters, polyetherketones,polyethersulfones, polyimides or mixtures thereof.

The non-reflective layer on the walls of the ZMW is generallytransparent to the light at the wavelength at which the ZMWs are used,so can also be referred to as non-opaque materials. While generallytransparent, the non-reflective layers need not be completelytransparent, and could be, for instance translucent. The non-reflectivelayer can be made of any suitable material that is generally transparentto the light used with the ZMW. The non-reflective layer material can beinorganic or organic. In some cases, the non-reflective layer comprisesan oxide or a nitride. Suitable oxides include oxides of aluminum,titanium, zinc, chromium, nickel, molybdenum, silver, magnesium, cesium,hafnium, zirconium, and silicon. In some cases, oxides of aluminum areused. As described in more detail below, in some cases thenon-reflective layer comprises an oxide of a metal which comprises theopaque cladding layer. Sol-gel materials can be used to form thenon-reflective layer, often composed largely of silicon oxides withsmaller amounts of other oxides. Polymeric materials can comprise thenon-reflective layer. Such polymers can be either largely carbon basedor silicon based. Suitable polymers include acrylates, methacrylates,polyimides, polyamides, polyketones, polysulfones, polyesters, cellulosebased polymers, polycarbonates, cyclic olefin polymers, sytrenicpolymers, fluorine-containing polymers, polyetherketones,polyethersulfones, polydimethylsiloxane (PDMS), poly(methylmethacrylate) (PMMA), and the like. Mixtures and copolymers of the abovepolymers can also be used.

As described above, it can be advantageous that the non-reflective layerhave a higher refractive index than that of the medium in the solutionvolume of the ZMW. We have shown by optical modeling that by having ahigher refractive index than the medium in the solution volume, a higherproportion of illumination light is directed to the solution volume thanif the non-reflective layer had the same or a lower refractive indexthan the medium in the solution volume. In many cases, an aqueous mediumis in the solution volume during analysis, generally having a refractiveindex of about 1.3. While it is beneficial to have a refractive indexgreater than the medium within the solution volume, it is notnecessarily advantageous to have the highest refractive index possiblein order, for example, to control hot spots. In some cases, thenon-reflective material has a refractive index of between about 1.4 andabout 5.0, between about 1.5 and about 3.0. In some cases, thenon-reflective material has a refractive index between about 1.7 andabout 2.6.

The ZMW's of the invention are particularly useful when employed as a anarray of ZMW's, allowing for parallel analysis of multiple luminescentspecies at one time; for example, multiple nucleic acid sequencingreactions.

The overall size of the array can generally range from a few nanometersto a few millimeters in thickness, and from a few millimeters to 50centimeters in width and/or length. Arrays may have an overall size ofabout few hundred microns to a few millimeters in thickness and may haveany width or length depending on the number of optical confinementsdesired.

The spacing between the individual confinements can be adjusted tosupport the particular application in which the subject array is to beemployed. For instance, if the intended application requires adark-field illumination of the array without or with a low level ofdiffractive scattering of incident wavelength from the opticalconfinements, then the individual confinements may be placed close toeach other relative to the incident wavelength.

Where the substrates comprise arrays of optical confinements, the arraysmay comprise a single row or a plurality of rows of optical confinementon the surface of a substrate, where when a plurality of lanes arepresent, the number of lanes will usually be at least 2, more commonlymore than 10, and more commonly more than 100. The subject array ofoptical confinements may align horizontally or diagonally long thex-axis or the y-axis of the substrate. The individual confinements canbe arrayed in any format across or over the surface of the substrate,such as in rows and columns so as to form a grid, or to form a circular,elliptical, oval, conical, rectangular, triangular, or polyhedralpattern. To minimize the nearest-neighbor distance between adjacentoptical confinements, a hexagonal array is sometimes preferred. Thearray need not be in rows or columns, and can be placed in any arbitrarypattern.

The array of optical confinements may be incorporated into a structurethat provides for ease of analysis, high throughput, or otheradvantages, such as in a microtiter plate and the like. Such setup isalso referred to herein as an “array of arrays.” For example, thesubject arrays can be incorporated into another array such as microtiterplate wherein each micro well of the plate contains a subject array ofoptical confinements.

In accordance with the invention, arrays of confinements or zero modewaveguides, are provided in arrays of more than 100, more than 1000,more than 10,000, more that 100,000, or more than 1,000,000 separatewaveguides on a single substrate. In addition, the waveguide arraystypically comprise a relatively high density of waveguides on thesurface of the substrate. Such high density typically includeswaveguides present at a density of greater than 10 zero mode waveguidesper mm², preferably, greater than 100 waveguides per mm² of substratesurface area, and more preferably, greater than 500 or even 1000waveguides per mm² and in many cases up to or greater than 100,000waveguides per mm². Although in many cases, the waveguides in the arrayare spaced in a regular pattern, e.g., in 2, 5, 10, 25, 50 or 100 ormore rows and/or columns of regularly spaced waveguides in a givenarray, in certain preferred cases, there are advantages to providing theorganization of waveguides in an array deviating from a standard rowand/or column format. In preferred aspects, the substrates include zeromode waveguides as the optical confinements to define the discretereaction regions on the substrate.

The ZMW arrays of the invention can be incorporated into substrateshaving micromirror structures, for example having one micromirror perZMW array for more effectively guiding the illumination light to thearray and/or guiding the emitted light from the array to a detector.Such micromirror structures are described, for example, in U.S.Provisional Patent Application 61/223,628, filed Jul. 7, 2009.

Apparatus/System

The invention also provides for systems and for apparatus that are usedin conjunction with the ZMW's and ZMW arrays of the invention to providereal-time analytical information on optical systems having luminescentspecies down to the single-molecule level. In particular, such systemstypically include the reagent systems, in conjunction with an analyticalsystem, e.g., for detecting data from those reagent systems. In certainpreferred embodiments, analytical reactions are monitored using anoptical system capable of detecting and/or monitoring interactionsbetween reactants at the single-molecule level. For example, such anoptical system can achieve these functions by first generating andtransmitting an incident wavelength to the reactants, followed bycollecting and analyzing the optical signals from the reactants. Suchsystems typically employ an optical train that directs signals from aplurality of reactions disposed on a solid surface having an array ofZMW structures of the invention onto different locations of anarray-based detector to simultaneously detect multiple different opticalsignals from each of multiple different reactions. The optical trainscan include optical gratings or wedge prisms to simultaneously directand separate signals having differing spectral characteristics from eachconfinement in an array to different locations on an array baseddetector, e.g., a CCD, and may also comprise additional opticaltransmission elements and optical reflection elements.

One aspect of the invention comprises an apparatus for obtaining nucleicacid sequence information comprising: a zero-mode waveguide arraystructure with a transparent substrate having a top surface, and areflective layer disposed upon the top surface of the transparentsubstrate; an array of apertures extending through the reflective layerto the transparent substrate wherein the apertures comprise wells havingwalls and bases, the bases of the wells comprising portions of the topsurface of the transparent layer; and a non-reflective layer disposed onthe walls of the wells wherein the thickness of the non-reflective layeris greater than about 5 nm. The zero-mode waveguide structure isincorporated into a device configured to hold an analysis solution incontact with the zero-mode waveguide structure, whereby the wellscomprise the analysis solution which comprises reagents for carrying outreactions for which nucleic acid sequence information can be derived;including polymerase enzyme, nucleotides, and nucleic acid template, thesolution further comprising fluorescent species. The system has: anillumination system that illuminates the wells through the transparentlayer; a detection system that detects emitted light over time from thefluorescent species within the wells, wherein the emitted light passesthrough the transparent layer; and a computing system that analyzes theemitted light over time in order to obtain sequence information.

An optical system applicable for use with the present inventionpreferably comprises at least an excitation source and a photondetector. The excitation source generates and transmits incident lightused to optically excite the reactants in the reaction. Depending on theintended application, the source of the incident light can be a laser,laser diode, a light-emitting diode (LED), a ultra-violet light bulb,and/or a white light source. Further, the excitation light may beevanescent light, e.g., as in total internal reflection microscopy,certain types of waveguides that carry light to a reaction site (see,e.g., U.S. Application Pub. Nos. 20080128627, 20080152281, and200801552280), or zero mode waveguides, described below. Where desired,more than one source can be employed simultaneously. The use of multiplesources is particularly desirable in applications that employ multipledifferent reagent compounds having differing excitation spectra,consequently allowing detection of more than one fluorescent signal totrack the interactions of more than one or one type of moleculessimultaneously. A wide variety of photon detectors or detector arraysare available in the art. Representative detectors include but are notlimited to an optical reader, a high-efficiency photon detection system,a photodiode (e.g. avalanche photo diodes (APD)), a camera, acharge-coupled device (CCD), an electron-multiplying charge-coupleddevice (EMCCD), an intensified charge coupled device (ICCD), and aconfocal microscope equipped with any of the foregoing detectors. Forexample, in some embodiments an optical train includes a fluorescencemicroscope capable of resolving fluorescent signals from individualsequencing complexes. Where desired, the subject arrays of opticalconfinements contain various alignment aides or keys to facilitate aproper spatial placement of the optical confinement and the excitationsources, the photon detectors, or the optical train as described below.

The subject optical system may also include an optical train which canhave multiple functions and may comprise one or more opticaltransmission or reflection elements. Such optical trains preferablyencompass a variety of optical devices that channel light from onelocation to another in either an altered or unaltered state. First, theoptical train collects and/or directs the incident wavelength to thereaction site comprising a ZMW structure of the invention. Second, theoptical train transmits and/or directs the optical signals emitted fromthe reactants to the photon detector. Third, the optical train mayselect and/or modify the optical properties of the incident wavelengthsor the emitted wavelengths from the reactants. Illustrative examples ofsuch optical transmission or reflection elements are diffractiongratings, arrayed waveguide gratings (AWG), optical fibers, opticalswitches, mirrors (including dichroic mirrors), lenses (includingmicrolenses, nanolenses, objective lenses, imaging lenses, and thelike), collimators, optical attenuators, filters (e.g., polarization ordichroic filters), prisms, wavelength filters (low-pass, band-pass, orhigh-pass), planar waveguides, wave-plates, delay lines, and any otherdevices that guide the transmission of light through proper refractiveindices and geometries. One example of a preferred optical train isdescribed in U.S. Patent Pub. No. 20070036511, filed Aug. 11, 2005, andincorporated by reference herein in its entirety for all purposes.

The reaction site, comprising a ZMW structure of the invention,containing a reaction of interest can be operatively coupled to a photondetector. The reaction site and the respective detector can be spatiallyaligned (e.g., 1:1 mapping) to permit an efficient collection of opticalsignals from the reactants. In certain preferred embodiments, a reactionsubstrate is disposed upon a translation stage, which is typicallycoupled to appropriate robotics to provide lateral translation of thesubstrate in two dimensions over a fixed optical train. Alternativeembodiments could couple the translation system to the optical train tomove that aspect of the system relative to the substrate. For example, atranslation stage provides a means of removing a reaction substrate (ora portion thereof) out of the path of illumination to create anon-illuminated period for the reaction substrate (or a portionthereof), and returning the substrate at a later time to initiate asubsequent illuminated period. An exemplary embodiment is provided inU.S. Patent Pub. No. 20070161017, filed Dec. 1, 2006.

Each individual reaction region can be operatively coupled to arespective microlens or a nanolens, preferably spatially aligned tooptimize the signal collection efficiency. Alternatively, a combinationof an objective lens, a spectral filter set or prism for resolvingsignals of different wavelengths, and an imaging lens can be used in anoptical train, to direct optical signals from each confinement to anarray detector, e.g., a CCD, and concurrently separate signals from eachdifferent confinement into multiple constituent signal elements, e.g.,different wavelength spectra, that correspond to different reactionevents occurring within each confinement. In some embodiments, the setupfurther comprises means to control illumination of each confinement, andsuch means may be a feature of the optical system or may be foundelsewhere is the system, e.g., as a mask positioned over an array ofconfinements. Detailed descriptions of such optical systems areprovided, e.g., in U.S. Patent Pub. No. 20060063264, filed Sep. 16,2005, which is incorporated herein by reference in its entirety for allpurposes.

The systems or apparatus of the invention also typically includeinformation processors or computers operably coupled to the detectionportions of the systems, in order to store the signal data obtained fromthe detector(s) on a computer readable medium, e.g., hard disk, CD, DVDor other optical medium, flash memory device, or the like. For purposesof this aspect of the invention, such operable connection provides forthe electronic transfer of data from the detection system to theprocessor for subsequent analysis and conversion. Operable connectionsmay be accomplished through any of a variety of well known computernetworking or connecting methods, e.g., Firewire®, USB connections,wireless connections, WAN or LAN connections, or other connections thatpreferably include high data transfer rates. The computers alsotypically include software that analyzes the raw signal data, identifiessignal pulses that are likely associated with incorporation events, andidentifies bases incorporated during the sequencing reaction, in orderto convert or transform the raw signal data into user interpretablesequence data (see, e.g., Published U.S. Patent Application No.2009-0024331, the full disclosure of which is incorporated herein byreference in its entirety for all purposes).

Exemplary systems are described in detail in, e.g., U.S. patentapplication Ser. No. 11/901,273, filed Sep. 14, 2007 and U.S. patentapplication Ser. No. 12/134,186, filed Jun. 5, 2008, the fulldisclosures of which are incorporated herein by reference in theirentirety for all purposes.

Further, the invention provides data processing systems for transformingsequence read data into consensus sequence data. The data processingsystems can include software and algorithm implementations to transformredundant sequence read data into consensus sequence data.

Various methods and algorithms for data transformation employ dataanalysis techniques that are familiar in a number of technical fields,and are generally referred to herein as statistical analysis. Forclarity of description, details of known techniques are not providedherein. These techniques are discussed in a number of availablereference works, such as those provided in U.S. Patent Publication No.20090024331 and U.S. Ser. No. 61/116,439, filed Nov. 20, 2008, thedisclosures of which are incorporated herein by reference in theirentireties for all purposes.

The software and algorithm implementations provided herein arepreferably machine-implemented methods, e.g., carried out on a machinecomprising computer-readable medium configured to carry out variousaspects of the methods herein. For example, the computer-readable mediumpreferably comprises at least one or more of the following: a) a userinterface; b) memory for storing redundant sequence read data; c) memorystoring software-implemented instructions for carrying out thealgorithms for transforming redundant sequence read data into consensussequence data; d) a processor for executing the instructions; e)software for recording the results of the transformation into memory;and f) memory for recordation and storage of the resulting consensussequence read data. In preferred embodiments, the user interface is usedby the practitioner to manage various aspects of the machine, e.g., todirect the machine to carry out the various steps in the transformationof redundant sequence read data into consensus sequence data,recordation of the results of the transformation, and management of theconsensus sequence data stored in memory.

As such, the methods further comprise a transformation of thecomputer-readable medium by recordation of the redundant sequence readdata and/or the consensus sequence data generated by the methods.Further, the computer-readable medium may comprise software forproviding a graphical representation of the redundant sequence read dataand/or the consensus sequence read data, and the graphicalrepresentation may be provided, e.g., in soft-copy (e.g., on anelectronic display) and/or hard-copy (e.g., on a print-out) form.

The invention also provides a computer program product comprising acomputer-readable medium having a computer-readable program codeembodied therein, the computer readable program code adapted toimplement one or more of the methods described herein, and optionallyalso providing storage for the results of the methods of the invention.In certain preferred embodiments, the computer program product comprisesthe computer-readable medium described above.

In another aspect, the invention provides data processing systems fortransforming sequence read data from one or more sequencing reactionsinto consensus sequence data representative of an actual sequence of oneor more template nucleic acids analyzed in the one or more sequencingreactions. Such data processing systems typically comprise a computerprocessor for processing the sequence read data according to the stepsand methods described herein, and computer usable medium for storage ofthe initial sequence read data and/or the results of one or more stepsof the transformation (e.g., the consensus sequence data), such as thecomputer-readable medium described above.

As shown in FIG. 8, the system 800 includes a substrate 802 thatincludes a plurality of discrete sources of chromophore emissionsignals, e.g., an array of zero mode waveguides of the invention havinglayers of non-reflective material disposed on their walls 804. Anexcitation illumination source, e.g., laser 806, is provided in thesystem and is positioned to direct excitation radiation at the varioussignal sources. This is typically done by directing excitation radiationat or through appropriate optical components, e.g., dichroic 808 andobjective lens 810, that direct the excitation radiation at thesubstrate 802, and particularly the ZMW structures of the invention 804.Emitted signals from the ZMW structures 804 are then collected by theoptical components, e.g., objective 810, and passed through additionaloptical elements, e.g., dichroic 808, prism 812 and lens 814, until theyare directed to and impinge upon an optical detection system, e.g.,detector array 816. The signals are then detected by detector array 816,and the data from that detection is transmitted to an appropriate dataprocessing system, e.g., computer 818, where the data is subjected tointerpretation, analysis, and ultimately presented in a user readyformat, e.g., on display 820, or printout 822, from printer 824. As willbe appreciated, a variety of modifications may be made to such systems,including, for example, the use of multiplexing components to directmultiple discrete beams at different locations on the substrate, the useof spatial filter components, such as confocal masks, to filter out-offocus components, beam shaping elements to modify the spot configurationincident upon the substrates, and the like (See, e.g., Published U.S.Patent Application Nos. 2007/0036511 and 2007/095119, and U.S. patentapplication Ser. No. 11/901,273, all of which are incorporated herein byreference in their entireties for all purposes).

One aspect of the invention is an apparatus for obtaining nucleic acidsequence information comprising a zero-mode waveguide array structurecomprising: a transparent substrate having a top surface, and areflective layer disposed upon the top surface of the transparentsubstrate; an array of apertures extending through the reflective layerto the transparent substrate wherein the apertures comprise wells havingwalls and bases, the bases of the wells comprising portions of the topsurface of the transparent layer; and a non-reflective layer disposed onthe walls of the wells wherein the thickness of the non-reflective layeris greater than about 5 nm. The zero-mode waveguide structure isincorporated into a device configured to hold an analysis solution incontact with the zero-mode waveguide structure, whereby the wellscomprise the analysis solution which comprises reagents for carrying outreactions for which nucleic acid sequence information can be derived;including polymerase enzyme, nucleotides, and nucleic acid template, thesolution further comprising fluorescent species. The apparatus furthercomprises an illumination system that illuminates the wells through thetransparent layer; a detection system that detects emitted light overtime from the fluorescent species within the wells, wherein the emittedlight passes through the transparent layer; and a computing system thatanalyzes the emitted light over time in order to obtain sequenceinformation.

Methods of Making ZMWs Having Non-Reflective Layers

The ZMW structures of the invention can be made using a number ofapproaches. In some cases, the process can involve first producing astructure having a lower transparent layer and an upper a cladding layerwith holes or apertures extending through the cladding to thetransparent layer; and subsequently depositing a layer of non-reflectivematerial onto the walls. In some cases, the deposition of thenon-reflective material can be carried out specifically, such thatdeposition only occurs on the cladding layer and not on the transparentsubstrate. In other cases, a conformal coating can be applied to thewhole surface non-selectively. In some cases the non-selectively coatedsubstrate can have the portions of the non-reflective material over thetransparent substrate selectively removed. It can be advantageous tohave some or all of the transparent substrate substantially free ofnon-reflective material, which can allow, for example, for the selectivereaction of a functionalizing agent or coupling agent to the surface ofthe transparent substrate. Such a selectively functionalized transparentsubstrate can be used to selectively bind a molecule of interest, suchas a polymerase enzyme selectively to the base of the ZMW structure.Such selective functionalization is described, for example in U.S.patent application Ser. No. 11/731,748, filed Mar. 29, 2007.

We have found that one useful method of forming the non-reflective layercomprises forming an oxide layer by controlled oxidation of materialthat constitutes the cladding layer. The oxide layer can be formed, forexample by thermal oxidation of the cladding layer in the presence ofoxygen and heat, or by electrochemical oxidation whereby the claddinglayer comprises an electrode. For example, where the cladding layercomprises aluminum, a layer of alumina can be formed on the surface ofthe aluminum by subjecting it to oxidizing conditions, either thermallyor electrochemistry. In some cases, an oxygen plasma is used to producethe oxide layer. Forming an oxide layer on the cladding has the benefitthat the non-reflective layer is formed selectively on the cladding, andis not formed on the transparent substrate.

One aspect of the invention is a method for forming a zero-modewaveguide structure comprising: providing a substrate having a lowertransparent layer and an upper metal layer, wherein the metal layercomprises an array of apertures disposed through the reflective layer tothe transparent layer, the apertures having side walls, and exposing thesubstrate to oxidizing conditions whereby an oxide layer is formed onthe side walls of the apertures under conditions whereby an oxide havinga thickness of greater than 5 nm is produced.

FIG. 9 illustrates processes for producing an oxide layer on the surfaceof the cladding layer to form the non-reflective layer of the invention.FIG. 9(A) shows the structure prior to oxidation to form thenon-reflective layer. The structure of FIG. 9(A), for example, a chip,has a transparent substrate 910 which has on its top surface an opaquecladding layer 920 which can be for example, a metal such as aluminum.The cladding layer has an array of apertures 930 extending through thecladding layer to the transparent substrate. The chip may have tens ofthousand to millions of apertures. This process may also be carried outon a wafer comprising many chips, which can later be separated by dicingof the wafer. In some cases, the cladding layer will have a thin 3 to 4nm native oxide on its surface. It is well known that metals such asaluminum will form a native oxide layer when exposed to air. This nativeoxide may or may not be removed prior to forming the non-reflectivelayer by oxidation.

The structure shown in 9(A) is exposed to oxidation conditions such asthermal (heat and oxygen), electrochemical, or oxygen plasma to form thestructure illustrated in FIG. 9(B) having non-reflective layer 940selectively on the cladding, and having substantially no non-reflectivelayer on the transparent substrate, except where it extends in from thecladding walls. Further oxidation results in a thicker non-reflectivelayer 950. As the oxidation proceeds, metal molecules on the surface ofthe cladding will generally become oxidized. Thus as the oxide layergrows, the ZMW aperture will generally become wider, and in addition,the metal portion of the cladding layer will become thinner due to theformation of oxide on the top surface of the cladding. At the same time,the cross sectional dimensions of the region inside the oxide layer (thesolution volume) will generally become smaller because each molecule ofoxide formed takes up more volume than that of the metal atom from whichthe oxide derives (e.g. Al₂O₃ has a higher molecular volume than Al₂).These dimensional changes can be taken into account in order to end upwith a ZMW having both the desired ZMW dimensions and the desiredsolution volume dimensions.

FIGS. 9(D) and 9(E) show the production of a non-reflective layer onto astructure in which the aperture 960 extends into the transparentsubstrate 910. Oxidation results in the formation of the non-reflectivelayer 970 which in some cases will grow to extend over the region of theaperture 960 that extends into the transparent layer.

Thermal oxidation is generally carried out in the presence of anoxidizing agent and water. Suitable thermal oxidation agents comprisechromates, cerates, permanganates, titanium or zirconium oxides, lithiumsalts, and molybdates. The temperature of the thermal oxidation isgenerally below 300° C.

Plasma oxidation is generally carried out by exposing oxygen and orozone at pressures from about 0.1 torr to about 100 torr, or betweenabout 1 torr and about 10 torr. The temperature can be from roomtemperature to about 400° C.

An alternative method for forming the non-reflective layer of theinvention is to deposit a layer onto a ZMW substrate. In some cases, thecoatings are deposited conformally in a manner that conforms to thetopography of the substrate. Coatings can, for example, be deposited inthe gas phase or in solution. Solution coating or deposition methods caninclude dipping, spraying, brushing, spin coating, meniscus coating,roller coating, curtain coating, extrusion coating, plasma deposited andelectrodeposition. In some cases, the coating will cover all surfacesincluding the transparent substrate at the bases of the ZMW apertures.In some cases, sol-gel chemistry can be used to produce, for example,silicon dioxide based non-reflective coatings. Chemical vapor deposition(CVD), for example with parylene, or plasma enhanced CVD (PECVD) oxidecan be used to form the non-reflective layer. In some cases, a selectiveAtomic Layer Deposition (ALD) process can be used to selectively form anon-reflective coating on the cladding walls. In other cases, an ALDprocess can be used to non-specifically coat the structure to form thenon-reflective layer, and the portion of the ALD layer over thetransparent layer can be subsequently removed where desired, for exampleusing etch-back. In some cases, Spin-on-Glasses (SOGs) can be spincoated onto the substrate to form the non-reflective layer, and theportion of the SOG over the transparent substrate can be subsequentlyremoved. These coating methods can deposit, for example, silicondioxide, silicon nitride, diamond, CVD oxide, CVD nitride, polymers suchas polyimides or teflon, and spin-on-glasses. In some embodiments, twoor more layers can be deposited as described above. For example, thenon-reflective layer can comprise a bi-oxide layer stack of Al₂O₃ andSiO₂, which can provide control of the etch-back process. Other coatingmethods, such as electrodeposition allow for selectively coating thenon-reflective layer on the cladding material. Materials and methodssuitable for deposition are described, for example in Franssila,“Introduction to Microfabrication”, Wiley, 2004, incorporated herein byreference for all purposes.

In some cases, electroplating or electrodeposition can be used todeposit a non-reflective layer. Generally, the part to be plated is thecathode of the circuit. Anode and cathode are immersed in an electrolytecontaining one or more dissolved salts as well as other ions to assistthe flow of electricity. A rectifier or battery supplies a directcurrent. At the cathode, the dissolved ions in the electrolyte solutionare reduced at the interface between the solution and the cathode, suchthat they “plate out” onto the cathode. In some cases, electrolessdeposition can be employed.

FIG. 10 illustrates a process of incorporating the non-reflective layeronto the walls of the ZMW by conformal coating. The structure in FIG.10(A) has a transparent substrate 1010 upon which a cladding layer 1020is disposed. The cladding layer 1020 has apertures 1030 extendingthrough the cladding layer to the transparent substrate. The structureof FIG. 10(A) is coated with the non-reflective layer material in aconformal manner such that the ZMW walls, the bases of the ZMWs, and thetop surface of the cladding layer is coated relatively uniformly. Theuse of such a conformal coating method has the advantage of beingstraightforward and manufacturable. However, the surface after thistreatment is uniform, precluding certain approaches toward specificallyfunctionalizing the base of the ZMW. In some cases, subsequent toconformal coating, the portion of the non-reflective layer over thetransparent substrate at the ZMW base can be selectively removed,exposing the transparent substrate at the ZMW base 1050, and allowingspecific functionalization of the surface, for example, using silanes.

FIG. 11 illustrates a process for producing non-reflective layers on theZMW walls where the ZMW aperture extends through the cladding layer andinto the transparent substrate. As above, the structure in FIG. 11(A)has a transparent substrate 1110 upon which a cladding layer 1120 isdisposed. The cladding layer 1120 has apertures 1130 extending throughthe cladding layer and extending into the transparent substrate. Thestructure of FIG. 11(A) is coated with the non-reflective layer materialin a conformal manner such that the ZMW walls, the bases of the ZMWs,and the top surface of the cladding layer is coated relativelyuniformly. Unlike the process shown in FIG. 10, here, since the apertureextends into the transparent substrate, the coating on the top of thetransparent substrate does not necessarily raise the base up into theZMW. In some cases, subsequent to conformal coating, the portion of thenon-reflective layer over the transparent substrate at the ZMW base canbe selectively removed, or etched back, exposing the transparentsubstrate at the ZMW base 1150, and allowing specific functionalizationof the surface, for example, using silanes. The etch-back step can beperformed, for example, by using photolithography to define the regionfor etch-back.

FIG. 12 illustrates a process similar to that of FIG. 11 but utilizing aplanar rather than a conformal coating. A planar coating 1240 is appliedto the ZMW array structure having apertures 1230 extending throughcladding layer 1220 to transparent layer 1210. The portion of thenon-reflective layer over the transparent substrate at the ZMW base canbe selectively removed, or etched back, exposing the transparentsubstrate at the ZMW base 1250, and allowing specific functionalizationof the surface, for example, using silanes. The etch-back step can beperformed, for example, by using photolithography to define the regionfor etch-back. Methods for forming the non-reflective layer also includemethods intermediate to that of FIG. 11 and FIG. 12, wherein asemi-conformal coating is applied, then subsequently selectivelyremoved, or etched back to produce the solution volume. The etch-backstep can be performed, for example, by using photolithography to definethe region for etch back.

Another method of preparing the non-reflective layers of the inventioncomprises growing polymeric layers from the surface of the claddingmaterial, for example by polymerization or by grafting. In oneembodiment an initiation monomer is attached to the surface of thecladding, either selectively on the walls or on the walls and topsurface. A source of monomer is then introduced, and polymerization fromthe walls is carried out. The thickness of the coating on the walls canbe controlled, by controlling the length (molecular weight) of thepolymer which is formed. Analogously, polymer can be grown from thesurface by attaching to the surface a catalyst, or catalyst/monomercomplex. Methods for controlling polymer molecular weight are well knownin the art. Methods for synthesizing polymers and controlling molecularweight are described, for example, in Braun et al., “Polymer Synthesis:Theory and Practice”, 4^(th) Edition, Springer, 2005. In otherembodiments, reactive groups on the surface of the cladding layer reactwith fully formed polymers to graft the polymers to the surface of thecladding. The thickness of the non-reflective layer produced in thismanner can be controlled by controlling the molecular weight of thepolymers attached to the cladding and the density of attachment.Selective polymerization and or grafting onto the cladding can also beaccomplished using electropolymerization.

Another method of preparing the non-reflective layers of the inventioncomprises depositing multilayers onto the surface, including depositingpolyelectrolyte multilayers. The multilayers can be selectivelydeposited on the cladding layer without being deposited on thetransparent substrate. For example, where the cladding layer is a metalsuch as aluminum, with a native aluminum oxide surface, and thetransparent layer comprises a silica material such as fused silica. Thedifference in reactivity of these surfaces can be used to facilitateselective deposition onto the cladding. In some cases, the walls of theZMWs can be treated in order to encourage multilayer deposition, and/orthe top of the cladding can be treated to discourage multilayerdeposition in order to allow for selective deposition on the walls ofthe ZMWs. The multilayers can be made to incorporate components thatincrease the refractive index in order to control the refractive indexof the non-reflective layer. Such components can include metal or metaloxide components including chromium, and titanium, or substituents suchas bromine. Polyelectrolyte multilayers can be conveniently formed, e.g.through successive deposition of alternating layers of polyelectrolytesof opposite charge. See, e.g., Decher (1997) Science 277:1232.

In one class of embodiments, a phosphate or phosphonate compound servesas the first layer on which a polyelectrolyte multilayer is built on thesurface, e.g., by successive deposition of oppositely chargedpolyelectrolytes.

The polymers comprising the multilayer can include phosphorouscontaining polymers such polymers comprising polyphosphonates. Aneffective approach to such multilayers uses multivalent cations toassemble the polyphosphonates containing polymers. Particularly usefulmultivalent cations are those of transition metals, and in particularthose of group IV transition metals, titanium (Ti), zirconium (Zr), orhafnium (Hf). A multilayer can be constructed with alternating layers.The multilayers can be produced by alternately treating the surface witha transition metal compound such as HfCl₂, ZrOCl₂, or ZrCl₄, and thenwith the phosphonate-containing polymer such as PVPA or Cp30.

In some cases, the transition metal compound and conditions can bechosen to have little or substantially no reactivity with SiO₂ such thatthe multilayer is selectively formed on the cladding layer.

The transition metal compounds can be introduced to the surface insolution, for example in water, methanol or a mixture of water andmethanol. The number of layers can be 2, 3, 4, 5, 6, 7, 8, 9, 10 or morethan 10 layers. In some embodiments 3 to 6 layers are used.

Electrochemical Oxidation—Anodization

We have found that one particularly useful method for producing thenon-reflective layers on the walls of the ZMWs is by electrochemicaloxidation (electrochemical anodization) of the cladding layer.Electrochemical anodization allows for the production of non-reflectivelayers on the ZMW in a controlled manner. The thickness of thenon-reflective layer can be controlled, for example, by controlling thevoltage, and or the current supplied during the electrochemical process.Where the conditions are controlled, a metal oxide non-reflective layercan be formed that is dense, robust, and smooth. We have found that therefractive index of the oxide layer that is formed is within a usefulrange for reducing illumination hot-spots, and for directing theillumination light into the solution volume of the ZMW.

One aspect of the invention is a method for forming a zero-modewaveguide array structure comprising: providing an electrochemicalsystem comprising a working electrode, a counter electrode, andoptionally a reference electrode; providing a substrate having a lowertransparent layer and an upper electrically conductive reflective layer,wherein the electrically conductive reflective layer comprises an arrayof apertures disposed through the reflective layer to the transparentlayer, the apertures having side walls, wherein the electricallyconductive reflective layer comprises the working electrode; andapplying a voltage to the working electrode such that a layer ofnon-reflective material is formed onto the side walls of the aperture.

The controlled electrochemical oxidation of the oxide layer can alsoprovide ZMW arrays having improved stability and longer life than ZMWsnot treated electrochemically. Arrays of optical confinements aresometimes used in environments which may cause the corrosion of metal ormetal oxide portions of the optical confinement structures. We havefound that even where measuring enzymes in optical confinement arraysunder conditions similar to biological conditions, corrosion of themetal or metal oxide portions of the arrays can negatively affectperformance. This may not be completely expected since the metals used,such as aluminum, are relatively robust, the biological conditions areoften at or near neutral pH, and the temperatures are relatively lowcompared to other industrial processes where metals such as aluminum areroutinely utilized. We have found, however, that corrosion of theoptical confinement array can negatively affect array performance evenwhen the metal surfaces that have been passivated withoutelectrochemical treatment. One reason why corrosion may become an issuewith optical confinement arrays where it is not an issue for similartypes of metal substrates is that changes in dimensions on the order ofnanometers to tens of nanometers can affect the performance of theoptical confinements. Such small dimensional changes may not be detectedin other uses. Another reason why the arrays of optical confinements mayexperience corrosion in these relatively mild environments is that theaqueous environments can, in some cases, have relatively high saltconcentrations. In addition to improving the corrosion resistance of theZMW array, the electrochemical treatment of the invention can alsoprovide for improved biochemical compatibility. Improved biochemicalcompatibility can include, for example, decreased deposition biologicalmolecules such as proteins, nucleotides, and nucleic acids onto thepassivated surfaces.

The electrochemical oxidation process of the invention is most usefulfor cladding layers which form stable oxide layers. One preferredmaterial for the cladding layer is aluminum. Other suitable materialswhich can form stable oxide layers include titanium, zinc, magnesium,and niobium. Alloys comprising these metals can also be used.

The oxidized layer is grown by passing a direct current through anelectrolytic solution, with the metal cladding, e.g. aluminum serving asthe anode (the positive electrode). The passage of current generallyreleases hydrogen at the cathode (the negative electrode) and oxygen atthe surface of the metal, e.g. aluminum anode, creating a layer ofaluminum oxide. Alternating current and pulsed current may also be used.The voltage may range from 1 to 300 V DC, from 5 to 100 V or from 10 to30 V. Conventional anodization processes are often carried out toencourage ongoing pitting of the growing oxide layer to avoid having theprocess terminated by auto-passivation. For the layers of oxide producedfor the ZMW's of the invention this type of re-pitting in order to growthicker layers is generally not required because of the relatively lowthickness of the coatings used (e.g. from 5 to 50 nm), and because ofthe desire for smooth and uniform coatings. Thus, in some cases, we havefound it desirable to allow for auto-passivation, for example, toprovide a fixed voltage between about 5 V and about 50 V or betweenabout 10V and about 30 V and allow the process to proceed untilauto-passivation significantly diminishes the current flow, thusdefining the thickness.

Pitting can also be encouraged by using highly acidic conditions. Wehave found that while conditions from about pH 2 to about pH 10 can beused, that more robust oxide layers can generally be formed by carryingout the process between about pH 4 and about pH 9, and in some casesbetween pH 4 and pH 6.

The electrochemical process of the invention can be carried out in anysuitable electrolyte. The preferred electrolyte is an electrolyte inwhich aluminum oxide is poorly soluble. Such electrolytes includeelectrolytes comprising, for example, borates, phosphates and tartrates.In some cases ammonium borate, phosphate, or tartrates are used. Anionshaving low mobility are generally preferred. In some cases, the presenceof mobile anions, such as chloride, fluoride and iodine can lead topitting, so the levels of such mobile anions is kept low, and in somecases the medium is substantially free of such ions. The electrolyte ischosen such that adequate control over thickness with voltage can beobtained. In some cases, the electrolytes allow the oxide growth to belinearly proportional to the applied potential. (J. Edwards Coating andSurface Treatment Systems for Metals. Finishing Publications Ltd. andASM International. pp. 34-38 (1997)).

We have found that arrays of optical confinements having metal layerscan be made more robust by providing an electrochemical treatment in thepresence of phosphorous containing compounds. In particular, we havefound that optical confinement arrays having a transparent layer and ametallic cladding layer have improved long term performance in solutionafter electrochemical passivation in the presence of phosphorouscontaining passivation compounds. The invention provides an anodizationprocess that creates, for example, a phosphonate-rich passivating oxidefilm on the metal, e.g. aluminum cladding. Anodization in the presenceof a phosphorous containing compound such as PVPA can result in theformation of a smooth oxidation layer as required for use with a zeromode waveguide application. In addition, these oxide coatings can berobust, with improved corrosion resistance. And, these coatings can alsoact as passivation layers with respect to biological contamination,assisting in the prevention of deposition of nucleic acids, nucleotides,proteins, and other biomolecules on the surface.

Electrochemical anodization of metals such as aluminum in the presenceof phosphorous containing passivation compounds can be implemented usingelectrochemical systems known in the art. The electrochemicalanodization can be carried out using a two electrode, or athree-electrode electrochemical cell having a counter electrode, anoptional reference electrode, and using the cladding layer of theoptical confinement array as the working electrode. The cell can beequipped with, for example, a graphite counter electrode and a saturatedcalomel reference electrode. The anodization can be achieved usingeither potentiostatic or cyclic deposition.

Suitable phosphorous containing electrochemical passivation compounds ofthe invention generally comprise P═O and/or P—OH functionality. Inparticular, compounds comprising phosphate or phosphonate groups can beused. Preferred passivation or coating compounds include phosphorouscontaining polymeric materials. Suitable phosphorous containingpolymeric materials include homopolymers and copolymers ofpoly(vinylphosphonic acid).

The phosphorous compounds for use in electrochemical depositions of theinvention can include the phosphorous containing selective passivationcompounds described herein.

These compounds will generally comprise P═O and/or P—OH functionality.In particular, compounds comprising phosphate or phosphonate groups canbe used. In some cases, these compounds will form strong bonds to ametal or metal oxide surface such as the surface of aluminum to providerobust passivation to the metal surface. Preferred passivation orcoating compounds include phosphorous containing polymeric materials.Suitable phosphorous containing polymeric materials include homopolymersand copolymers of poly(vinylphosphonic acid), Albritect™ CP-30,Albritect™ CP-10, Albritect™ CP-90, Aquarite® ESL, and Aquarite® EC4020.Albritect™ and Aquarite® compounds are commercially available fromRhodia, Inc. Phosphate or phosphonic acid moieties can in some casesbind strongly to metal oxides (e.g., aluminum oxide, titanium oxide,zirconium oxide, tantalum oxide, niobium oxide, iron oxide, and tinoxide) during the electrochemical process, forming phosphorouscontaining oxides. Thus, compounds that comprise at least one phosphategroup (—OP(O)(OH)₂, whether protonated, partially or completelydeprotonated, and/or partially or completely neutralized) or phosphonicacid group (—P(O)(OH)₂, whether protonated, partially or completelydeprotonated, and/or partially or completely neutralized) can be used.

The electrolyte can contain an alkyl phosphate or an alkyl phosphonate.The terms phosphonic acid and phosphonate are alternatively used torefer to the compounds described herein. It is understood that aphosphonic acid will generally have hydrogens associated with two of thephosphonic acid oxygens, and that a phosphonate will generally haveother counterions associated with these oxygens. In aqueous solution,hydrogen ions and counterions can exchange rapidly. Thus generallyeither phosphonic acid and phosphonate compounds can be useful in theinvention.

Exemplary alkyl phosphates and alkyl phosphonates include, but are notlimited to, an alkyl phosphate or alkyl phosphonate in which the alkylgroup is a straight chain unsubstituted alkyl group (e.g., a straightchain alkyl group having from 1 to 26 carbons, e.g., from 8 to 20carbons, e.g., from 12 to 18 carbons). Additional exemplary alkylphosphates and alkyl phosphonates include functionalized or substitutedalkyl phosphonates and alkyl phosphates, for example, functionalizedX-alkyl-phosphonates and X-alkyl-phosphates where X is a terminal groupcomprising or consisting of a vinyl (CH₂), methyl (CH₃), amine (NH₂),alcohol (CH₂OH), epoxide, acrylate, methacrylate, thiol, carboxylate,active ester (NHS-ester), melamine, halide, phosphonate, or phosphategroup, or an ethylene glycol (EG) oligomer (EG4, EG6, EG8) orpolyethylene glycol (PEG), photo-initiator (e.g., photo-inverters suchas dithiocarbamates (DTC)), photocaged group, or photoreactive group(e.g., psoralen). The alkyl chain spacer in the X-alkyl-phosphonate orX-alkyl-phosphate molecule is a hydrophobic tether that optionally has 1to 26 methylene (CH₂) repeat units, preferably from 8 to 20, and morepreferably from 12 to 18. The alkyl chain may contain one or more (up toall) fluorinated groups and/or can instead be a hydrocarbon chain withone or more double or triple bonds along the chain. TheX-alkyl-phosphate or X-alkyl-phosphonate layer can furthermore be usedas a substrate to anchor other ligands or components of the surfacestack, such as a polyelectrolyte multilayer or chemisorbed multilayer.The alkyl phosphates/phosphonates can form a stable, solvent resistantself-assembled monolayer that can protect the underlying material (e.g.,aluminum) from corrosion etc.; the role of the alkyl tether in the abovestructures is to enhance the lateral stability of the chemisorbedmonolayer in aqueous environments. In embodiments in which thephosphonate or phosphate compound includes an unsaturated hydrocarbonchain, the double or triple bond(s) can serve as lateral crosslinkingmoieties to stabilize a self-assembled monolayer comprising thecompound. Specific exemplary alkyl phosphates and alkyl phosphonatesinclude, but are not limited to, octyl phosphonic acid, decyl phosphonicacid, dodecyl phosphonic acid, hexadecyl phosphonic acid, octadecylphosphonic acid, docosyl phosphonic acid (i.e., C22 phosphonic acid),hydroxy-dodecyl phosphonic acid (HO(CH₂)₁₂P(O)(OH)₂),hydroxy-undecenyl-phosphonic acid, decanediylbis(phosphonic acid),dodecylphosphate, and hydroxy-dodecylphosphate.

Suitable phosphonates include high molecular weight polymericphosphonates such as polyvinylphosphonic acid (PVPA):

wherein n can be from about 1 to about 1000 or from about 10 to about100.

Phosphonate end-capped polymers of polymers having acidic functionalgroups such as carboxylic acids, sulfonic acids and mixtures thereof canalso be used. These can include phosphonate end-capped poly(acrylates),poly(sulfonates), and copolymers thereof. Exemplary phosphonateend-capped compounds include:

where n can be from about 1 to 1000, can be between about 10 and about100, and can be about 20 (available from Rhodia, Inc. as Aquarite®EC4020), or

where n and m can be from about 1 to about 1000, from about 10 to about100, or can each be about 50, in some cases, m is about 24 and n isabout 16 (available from Rhodia, Inc. as Aquarite® ESL). Exemplarycopolymers copolymer include the copolymers:

such as vinyl phosphonic acid-acrylic acid copolymers (commerciallyavailable from Rhodia as Albritect™ CP30). The values for n and m canrange from about 1 to about 1000. In some cases, m is between about 10and about 100, and n is between about 100 and 300. In some cases, m isbetween about 50 and about 70, and n is between about 80 and 120. Insome cases, m is about 60 and n is about 200.

Suitable phosphonates also include low molecular weight phosphonatessuch as 2-carboxyethyl phosphonic acid (also known as3-phosphonopropionic acid; commercially available from Rhodia asAlbritect™ PM2). Other suitable low molecular weight phosphorouscontaining compounds are described in U.S. patent application Ser. No.11/394,352.

One aspect of the invention is a method for producing azero-mode-waveguide array comprising: providing an electrochemical cellhaving a working electrode, a counter electrode, and optionally areference electrode, wherein the working electrode comprises a metallicupper layer of a substrate also having a transparent lower layer,wherein the metallic upper layer comprises an array of aperturesextending through metallic upper layer to the transparent lower layer;contacting the working electrode with a solution comprising aphosphorous containing compound; and passing current through theelectrochemical cell whereby a phosphorous containing material isdeposited onto the metallic upper layer of the substrate.

The electrochemical anodization is generally carried out in aqueoussolution. The phosphorous containing passivation compounds, e.g.phosphonate containing polymers, are generally provided inconcentrations from about 0.01% to about 20% (weight by volume (e.g.g/cc)). The temperature can be around room temperature. In some cases,temperatures from 20° C. to about 90° C. are used. While carrying outthe anodization, the current can be monitored in order to control theextent of the anodization/passivation reaction.

Electrochemical anodization methods of the invention can result inoptical confinement arrays having that exhibit less corrosion thanoptical confinement arrays that have not been electrochemicallypassivated. The electrochemically passivated arrays can have more than30%, 40%, 50%, 70% 80% or 90% lower rates of corrosion than arrays whichhave not been electrochemically passivated with the methods of theinvention. In some cases, the electrochemically passivated arrays canhave more than 2 times, 3 times, 4 times, 6 times, or 10 times lowerrates of corrosion than arrays which have not been electrochemicallypassivated with the methods of the invention.

Methods of Use

The ZMWs of the invention can be utilized for the optical measurement ofanalytes at very small amounts, down to the level of single molecules.By controlling the solution volume within a ZMW structure, higherquality measurements can be made of single molecule systems.

As described herein, the systems of the invention can be used foranalyzing molecules in very small volumes. The individual confinement inthe array can provide an effective observation volume less than about1000 zeptoliters, less than about 900, less than about 200, less thanabout 80, less than about 10 zeptoliters. The systems of the inventioncan provide for measurements at or near physiological conditions, forexample, in the range from micro-molar to millimolar.

One aspect of the invention is a method for analyzing a luminescentspecies comprising: disposing a luminescent species in an aperture thatextends through an upper reflective layer that is disposed on a lowertransparent layer, wherein the aperture comprises side walls, and anon-reflective layer on the side walls of the aperture having athickness of greater than 5 nm; and detecting emitted light from theluminescent species wherein the emitted light passes through thetransparent layer.

The systems can be used to measure optical properties of molecules usingluminescent indicators. Luminescent tags such as dyes and fluorescentnanoparticles can be incorporated into or near the analytical moleculesof interest. Fluorescent methods are particularly useful, includingusing dye-dye interactions such as Forster Resonance Energy Transfer(FRET) in order to measure molecular events for a single molecule.Observations in real-time can be made of (1) distributions andfluctuations in enzymatic activity, (2) reaction mechanisms, and (3)transient intermediates that are otherwise difficult to capture inconventional experiments due to their low steady state concentrations.

In certain aspects, the subject invention provides substrates andmethods for performing single-molecule observation. The optical arraysof the invention can provide information on individual molecules whoseproperties are hidden in the statistical mean that is recorded byordinary ensemble measurement techniques. In addition, because ofmultiplexing, the arrays are conducive to high-throughputimplementation, requiring small amounts of reagent(s), and takingadvantage of the high bandwidth of modern avalanche photodiodes forextremely rapid data collection. Moreover, because single-moleculecounting automatically generates a degree of immunity to illuminationand light collection fluctuations, single-molecule analysis can providegreater accuracy in measuring quantities of material than bulkfluorescence or light-scattering techniques. As such, the subjectsubstrates and devices may be used in a wide variety of circumstancesincluding sequencing individual human genomes as part of preventivemedicine, rapid hypothesis testing for genotype-phenotype associations,in vitro and in situ gene-expression profiling at all stages in thedevelopment of a multi-cellular organism, determining comprehensivemutation sets for individual clones and profiling in various diseases ordisease stages. Other applications involve profiling of cell receptordiversity, identifying known and new pathogens, exploring diversitytowards agricultural, environmental and therapeutic goals.

In preferred embodiments, the instant invention is directed to observingnucleic acid sequencing reactions, e.g., sequencing-by-incorporationreactions. In preferred embodiments, such an illuminated reactionanalyzes a single molecule to generate nucleotide sequence datapertaining to that single molecule. For example, a single nucleic acidtemplate may be subjected to a sequencing-by-incorporation reaction togenerate one or more sequence reads corresponding to the nucleotidesequence of the nucleic acid template. For a detailed discussion of suchsingle molecule sequencing, see, e.g., U.S. Pat. Nos. 6,056,661,6,917,726, 7,033,764, 7,052,847, 7,056,676, 7,170,050, 7,361,466,7,416,844; Published U.S. Patent Application Nos. 2007-0134128 and2003/0044781; and M. J. Levene, J. Korlach, S. W. Turner, M. Foquet, H.G. Craighead, W. W. Webb, SCIENCE 299:682-686, January 2003 Zero-ModeWaveguides for Single-Molecule Analysis at High Concentrations, all ofwhich are incorporated herein by reference in their entireties for allpurposes.

Methods for Obtaining High Loading of Single Molecules in ZMWs—Islandsof Functionalizing Agent

As described above, arrays ZMWs are useful for optically analyzingreactions of many single molecules simultaneously. When performing suchanalyses, it is generally desirable to maximize the number of ZMWs inthe array that are occupied with a single molecule of interest, and tominimize the number of ZMWs in the array that have zero or have morethan one single molecule of interest (e.g., template or other analyteand/or enzyme). Loading two or more molecules of interest into a ZMW orother small observation volume tends to complicate any analysis ofsignals observed from double (or more than double)-loaded region. Thisis because two (or more) sets of signals may simultaneously be observedfrom the ZMW or other observation volume, meaning that the signals fromthe ZMW would have to be deconvoluted before data from the observationregion could be used. More typically, data from double (+) loaded ZMWscan be recognized by various data analysis methods, and data frommis-loaded ZMWs or other relevant observation volumes is simplydiscarded.

To reduce the incidence of multiple molecule loading events in therelevant reaction/observation volume(s) of the array, it is typical inthe art to substantially “under-load” the array with the analytemolecules of interest. See, for example “Improved fabrication ofzero-mode waveguides for single-molecule detection” (2008) Foquet et al.Journal of Applied Physics 103, 034301. Random distribution of moleculesinto the array results in one or fewer molecules being loaded into mostreaction/observation volumes when fewer than 37% of all observationvolumes are loaded. This type of loading is referred to as“Poisson-limited” analyte loading, meaning that few enough molecules areadded to the array so that a Poisson-style random statisticaldistribution of the analytes into the array results in one or feweranalytes per observation volume in most cases. Some approaches toachieving single molecule loading above the Poisson distribution aredescribed in U.S. patent application Ser. No. 12/384,097 filed Mar. 30,2009.

One aspect of the invention is a method of obtaining an island offunctionality within a nanoscale aperture in a cladding layer on thesurface of a substrate. The method comprises growing a constrictionlayer selectively on the cladding layer in order to constrict thenanoscale aperture such that a portion of the substrate within theaperture is still exposed, and functionalizing the exposed portion ofthe substrate to provide reactive species on these exposed portions.Some or all of the constriction layer is removed, thereby producing anisland of functionalized surface within the nanoscale aperture at itsbase. The island of functionality can be used to couple a molecule ofinterest, such as a single molecule to the base of the aperture. Thecoupling of the single molecule can be performed before the removal ofthe constriction layer or after the removal of the constriction layer.The island of functionality can be used to attach a single particle orbead within each aperture. The single particle or bead can be inorganicor organic, it can comprise a polymer, and can comprise a metal or metaloxide. The particle can be, for example, a quantum dot.

An exemplary process of the invention for forming the island offunctionality is shown in FIG. 13. A substrate 1310 is provided whichhas a cladding layer 1320 on its top surface. The substrate can be, forexample a silicon substrate. In some cases, the substrate is atransparent material such as SiO2, and the cladding layer is an opaquematerial such a metal e.g. aluminum. The cladding layer has a pluralityof nanoscale apertures 1330 extending through the cladding layer,exposing portions of the top surface of the substrate 1310. In step I,an aperture constriction layer 1340 is selectively grown on the claddinglayer. As this constriction layer grows in from the walls of theaperture, it makes the aperture smaller, exposing a fraction of thesubstrate surface that was exposed prior to growing the constrictionlayer. After the growth of the constriction layer, in step II, theportion of the surface that is left exposed 1350 is treated with agentsthat react with the surface in order to deposit functional groups ontothe surface of the substrate. The deposition can be selective to thesubstrate surface as shown in FIG. 13, or the deposition can benon-selective, adding functional groups to both the substrate and theconstriction layer surfaces. In step III, the aperture constrictionlayer is removed to leave an island of functionality 1360 within theaperture that can be used for further selective coupling to the surfaceof the substrate.

In some aspects, the invention is directed toward a method for producingan island of functionalizing agent in an array of ZMW's comprising: a)providing a substrate having on its surface a cladding layer, whereinthe cladding layer comprises an array of apertures disposed through thecladding layer to the substrate, the apertures having side walls; b)selectively growing an aperture constriction layer on the cladding layersuch that the aperture constriction layer extends in from the side wallsof the aperture to reduce the cross-sectional dimensions of theaperture; c) attaching functionalizing agent to exposed regions of thesubstrate within the apertures; and d) removing the apertureconstriction layer whereby an array of apertures, each having an islandof functionalizing agent is produced.

In some cases, the aperture has a generally cylindrical profile and hasa diameter that is from about 70 nm to about 300 nm, or from about 90 nmto about 200 nm. In some cases, the island that is formed is from about5 nm to about 50 nm, or from about 10 nm to about 40 nm.

The constriction layer can be, for example a polymer such as an organicpolymer, a metal, or a metal oxide or nitride. The growth of theconstriction layer can be produced, for example, by polymerization,electrochemical processes such as electroplating or electrodeposition,by oxide growth, or by chemical vapor deposition (CVD).

In some embodiments the constriction layer comprises a polymer that isgrown from the side walls of the cladding. This can be accomplished byselectively depositing an initiator onto the cladding layer and thengrowing a polymer from the initiators using well known organic polymersynthesis chemistry. This can be accomplished, for example, bydepositing onto the cladding a co-polymer consisting of an initiator anda passivator. In some cases, the cladding is aluminum, and thepassivator comprises a polymeric component that selectively binds toaluminum. Such cladding selective polymeric components are described,for example in U.S. Patent Application Publication No. 2008/0032301filed Mar. 29, 2007. An example of a copolymer that comprises aninitiator and a passivator is poly(vinylphosphonicacid-co-hydroxyethylmethacrylate)

In some cases a degradable linker is used that has at one end a groupthat specifically attached to the cladding, in the middle a linker, andat the end an initiator or polymer growth site. Where the cladding is ametal such as aluminum, the group that attaches to the cladding can be aphosphate or phosphonate group. The linker can be any suitable spacergroup. The linker can include a degradable piece that allows forcleavage of within the linker to facilitate removal of the polymerconstriction layer. The degradable portion can be, for example, a groupthat is cleaved with light, heat, acid, base, or catalyst. The polymergrowth site is a site at which the polymer begins to grow after polymersynthesis is initiated. The polymer synthesis can be free-radical, e.g.azo initiated, or light activated. In some cases the linker can comprisea bidentate linker held together with an azo initiating group. Examplesof such linkers include compounds (I), (II), and (III) shown below.

Each of the phosphate groups can bind to the surface, producing twofree-radical initiation/polymerization sites upon activation. In somecases the linkers include hydrophobic portions such as chains ofmethylene groups from about 3 to about 20 in length can be useful.Compounds I and II can be made by forming the ester group, for exampleusing a carbodiimide coupling reaction. For example, compound I can beformed with a dicyclohexyl carbodiimide coupling in THF with in thepresence of DMAP from 6-phosphohexanoic acid and2,2′-Azobis[2-methyl-N-(2-hydroxyethyl)propionamide. In some cases, thephosphate groups are protected, e.g. as methyl groups prior to thecoupling reaction. In some cases, the groups can be made by forming theamide bonds in the chain using carbodiimide coupling.

The chemistry used to synthesize the polymers from the walls can be anysuitable polymerization chemistry. For example, the polymers can begrown either through a random graph polymerization (RGP) or via a livingradical polymerization. The polymers can be grown, for example, usingring opening polymerization (ROP) or light-initiated polymerization. Thepolymer can be a copolymer such as poly(vinylphosphonicacid-co-hydroxyethylmethacrylate). The thickness of the constrictionlayer can be controlled by controlling the length of the polymermolecules using polymerization conditions. Time, temperature, type ofsolvent, and concentrations of reaction components can be used tocontrol the polymerization rate. Where the polymerization solventcomprises water, the pH, the level and type of cosolvent, and the leveland type of ions in solution can also be used to control the extent ofreaction, thereby controlling the thickness of the polymer on the walls.There will generally be a range of lengths of polymer generated by thepolymerization procedure. It is generally desired to choose conditionsthat provide a relatively narrow range of polymer lengths. This can beaccomplished in part by selecting polymerization chemistry that tends tohave a low level of polydispersity.

Once the polymer constriction layer is produced, the open portion of thesubstrate, for example a transparent substrate such as fused silica istreated with reagents to produce a functionalized surface. Thefunctionalized surface will generally comprise coupling groups forattaching single molecules or single particles. The polymericconstriction layer is then removed from the walls, leaving an island offunctional groups. The functional groups can comprise one or moresilanes, for example functionalized silanes, such that the silaneportion of the molecule can couple to the surface allowing thefunctional part of the silane to act as a coupling group. In some cases,the molecule or particle of interest is attached to the island offunctional groups before removal of the polymer, and in other cases, thefunctional groups after the polymeric constriction layer is removed. Theremoval of the polymers can be accomplished in a variety of waysdepending on the type of polymer that is used to coat the walls. Heat,light, solvents, acids, bases, and catalysts can be used to remove thepolymer. The polymers can be designed to have groups along the backbonethat will break down, fragmenting the polymer into smaller pieces tofacilitate removal Examples of polymers to use in the constriction layerthat are degradable by acid or hot water are poly(lactic acid) andpoly(caprolactone). See e.g. Yang et al. “Surface-Initiated Ring-Openingpolymerization of ε-Caprolactone from the surface of PP Film”, J. appl.Poly. Sci., 2007, 105, 877-884.

In some cases, after removal of the constriction layer, the portions ofthe substrate exposed by removal of the constriction layer are treatedwith a passivating compound such as a silane in order to preventunwanted binding of other components. This passivation can be carriedout, for example, in cases where the molecule or particle of interest isattached after removal of the constriction layer. The passivation can beperformed to prevent unwanted binding of the molecule or particle ofinterest to the exposed portions of the surface.

The constriction layer can be formed by electroplating orelectrodepositing onto the surface of the cladding. This can be useful,for example, when the cladding comprises a conductive material such as ametal. Electrodeposition is a process of producing a coating, eithermetallic or non-metallic, on a surface by the action of electriccurrent. The deposition of a metallic coating onto an object isgenerally achieved by putting a negative charge on the object to becoated and immersing it into a solution which contains a salt of themetal to be deposited (i.e., the object to be plated is made the cathodeof an electrolytic cell). The metallic ions of the salt carry a positivecharge and are thus attracted to the object. When they reach thenegatively charged cladding, the cladding provides the current requiredto reduce the positively charged ions to metallic form. The metal thatis plated is different than the cladding to allow for subsequent removalwithout removing the cladding. Electrodeposited metals include copper,nickel, gold, silver, chrome, cadmium, bronze, rhodium, black nickel,and zinc. In some cases non-metals can be electrodeposited as theconstriction layer. In such cases, the cladding can be the anode or thecathode of the electrochemical system. Electrodeposition processesuseful in the invention are described, for example, in Paunovic et al.“Fundamentals of Electrochemical Deposition”, Wiley, New York, 1998, thecontents of which are incorporated by reference herein for all purposes.

One aspect of the invention comprises using the oxidation methods of theinvention in order to produce an island of functionality within the ZMWand to achieve high loadings of the single molecules of interest. Themethods can be used, for example, to obtain higher loadings of singlemolecules than can be achieved through Poisson-limited analyte loading.Higher loadings of single molecules can be obtained by having a smallregion (an island) of functional groups or coupling agent within theZMW.

An aspect of the invention is a method for obtaining an island offunctionality at the bases of an array of ZMWs comprising: providing anelectrochemical system comprising a working electrode, a counterelectrode, and optionally a reference electrode; providing a substratehaving a lower transparent layer and an upper cladding layer, whereinthe cladding layer comprises an array of apertures disposed through thereflective layer to the transparent layer, the apertures having sidewalls, wherein the cladding layer comprises the working electrode;applying a voltage to the working electrode such that a layer of oxideis formed onto the side walls of the aperture, attaching functionalizingagent to exposed regions of the transparent layer within the apertures;and dissolving the oxide layer from the walls of the aperture wherebyislands of functionalizing agent are formed within the apertures.

FIG. 14 show an illustration of an embodiment of the invention in thatproduces islands of functionalizing agent within a ZMW by growing anoxide layer onto the cladding. FIG. 14(A) shows a portion of a substratehaving an array of apertures 1430 through cladding layer 1420 totransparent layer 1410. In step I, an oxide layer 1440 is grown ontocladding layer 1420. The oxide can be grown thermally, with plasmatreatment, or electrochemically. In order to grow the oxideelectrochemically, for example, the cladding layer is attached to apower supply such that the cladding can act as the working electrode inan electrochemical system. The working electrode and a counter electrodeare placed into an electrolyte solution and the electrochemicaloxidation is carried out such that an oxide layer 1440 is formed fromthe cladding layer. As described above, the oxidation process generallyresults both in an oxide layer extending out from the walls, and also inthe oxide layer extending into the cladding layer as the claddingmaterial is consumed to form the oxide layer. Therefore the oxideextends into the ZMW and the ZMW cross-section becomes larger. Afterstep I, a shown in FIG. 14(B), a smaller portion of the transparentsubstrate is now exposed than before the process. In step II, afunctionalizing agent 1450 is attached to the transparent surface. Forthe embodiment shown in FIG. 14(C), the functionalizing agent isattached using a selective process such that little or nofunctionalizing agent becomes attached to the oxide layer on the wallsof the ZMW. In other embodiments, functionalizing agent that is lessselective or that is completely non selective can be used.Functionalizing agent attached to the walls will tend to be remove instep III of the process. In step III, the oxide is dissolved, forexample, using acidic aqueous solution. The removal of the oxide fromthe walls produces a ZMW structure having an island of functionalizingagent 1460 within the ZMW.

Attachment of functional agents to the transparent materials may becarried out by any of a variety of methods known in the art. Forexample, in the context of silica based substrates, e.g., glass, quartz,fused silica, silicon, or the like, well characterized silanechemistries may be used to couple other agents to the surface. Suchother agents may include functional groups, activatable groups, and/orlinker molecules to either of the foregoing, or the actual molecules ofinterest that are intended for use in the end application of thesurface. In some cases the functional groups comprise coupling groupsfor coupling a molecule of interest or an intermediate to the surface.In the context of other transparent material types, e.g., polymericmaterials, or the like, other processes may be employed, e.g., usinghybrid polymer surfaces having functional groups coupled thereto orextending from the polymer surface using, e.g., copolymers withfunctional groups coupled thereto, metal associative groups, i.e.,chelators, thiols, or the like.

Where the transparent material comprises a silica-based surface, silanes(e.g., methoxy-, or ethoxy-, silane reagents) can form stable bonds withsilica surfaces via Si—O—Si bond formation, and are less reactive tometal or metal oxide surfaces such as aluminum or aluminum oxidesurfaces under appropriately selected reaction conditions (e.g., vaporphase, solution-based treatments). Silanes, for example, silanesmodified with coupling groups for attachment of enzymes or othermolecules of interest (e.g., biotin-PEG-silanes such as those describedin U.S. patent application Ser. No. 11/240,662), can thus be used tobind desired molecules to silica surfaces such as those in a ZMW.

In some cases, the coupling groups are activatable or deactivatablecoupling groups. A variety of different activatable or deactivatablecoupling groups may be used in conjunction with this aspect of theinvention. Typically, such groups include coupling groups that arecapped or blocked with a selectively removable group. These includegroups that are thermally altered, e.g., thermolabile protecting groups,chemically altered groups, e.g., acid or base labile protecting groups,and photo alterable groups, e.g., photo-cleavable or removableprotecting groups. Suitable activatable and deactivatable couplinggroups are provided, for example, in U.S. patent application Ser. No.11/394,352.

A variety of different coupling groups may be used in this context,depending upon the nature of the molecule of interest to be subsequentlydeposited upon and coupled to the substrate. For example, the couplinggroups may include functional chemical moieties, such as amine groups,carboxyl groups, hydroxyl groups, sulfhydryl groups, metals, chelators,and the like. Alternatively or additionally, they may include specificbinding elements, such as biotin, avidin, streptavidin, neutravidin,lectins or SNAP-Tags™ and their substrates (Covalys Biosciences AG; theSNAP-Tag™ is a polypeptide based on mammalianO6-alkylguanine-DNA-alkyltransferase, and SNAP-tag substrates arederivates of benzyl purines and pyrimidines), associative or bindingpeptides or proteins, antibodies or antibody fragments, nucleic acids ornucleic acid analogs, or the like. Click chemistry including theAzide-Alkyne Huisgen Cycloaddition catalyzed, for example, by copper canalso be used.

Additionally, or alternatively, the coupling group may be used to couplean additional group that is used to couple or bind with the molecule ofinterest, which may, in some cases include both chemical functionalgroups and specific binding elements. A preferred set of embodimentsutilizes biotin to attach a molecule of interest to the silica-based ortransparent substrate. The attachment of biotin or other selectivebinding group to the surface can be accomplished in a number of ways.

One exemplary approach involves reacting a silica-based surface regionwith a compound having a silane group directly coupled to the selectivebinding group, for example, a silane-polyethylene glycol-biotin compoundto produce a surface having selective binding groups, e.g. biotin boundto the silica-based region. This method provides a one step process forobtaining a silica-based surface having selective binding groups such asbiotin attached thereto. In some cases, the silane compound having theselective binding group is diluted with a silane that does not containthe selective binding group, e.g. silane-polyethylene glycol in order tocontrol the density of selective binding groups on the silica-basedsurface.

Another exemplary approach involves first reacting a silica-basedsurface with a coupling agent, and reacting the coupling agent on thesurface with an attaching agent that has both functionality for reactingwith the coupling agent, and functionality for attaching the desiredmolecule (e.g. a selective binding agent such as. Biotin). For example,the silica-based surface is reacted with an aminosilane or thiol-silaneunder conditions where the aminosilane or thiol-silane becomes bound tothe substrate. The aminosilane or thiol-silane surface is subsequentlyreacted with an attaching agent, for example having an activated estercoupled to biotin to link the biotin to the aminosilane surface, or amaleimide group coupled to biotin to link to the thiol-silane surface.The attaching agent can be diluted as described herein with moleculesthat react, for example, with the aminosilane or thiol-silane, but donot have selective binding groups. This process incorporating anattaching group results in the coupling the selective binding agent tothe surface in two steps. While this approach uses two steps rather thanthe one step described above, it can have some advantages indevelopment, processing, and quality control.

The linking chemistry between the coupling agent and the compound havingthe selective binding agent can comprise any suitable linking chemistry.The linking chemistry can comprise, for example, thiol-maleimide,anhydride-amine, alkyne-azide, epoxide-amine, or amine-activated ester.As with the one step method, the compound having the selective bindingagent can be diluted with a compound with the same reactivefunctionality, but not having the selective binding agent to control thedensity of selective binding agent on the surface.

In some cases, the compound comprising the selective binding agent isnot diluted with another agent such as a capping agent that can bind tothe surface, but does not have selective binding agent. Where there isno dilution, a relatively highly density of selective binding agent canbe achieved. This high level of selective binding agent allows eitherfor attaching a relatively high density of molecules of interest to thesurface, or can be used to attach relatively few molecules of interestto the surface.

In some cases, the compound comprising the selective binding agent isdiluted with another agent such as a capping agent that can bind to thesurface, but does not have selective binding agent. In accordance withthe invention, the low density of the coupling agent on a surface isdesigned to provide a single reactive moiety within a relatively largearea for use in certain applications, e.g., single molecule analyses,while the remainder of the area is substantially non-reactive. As such,coupling groups can be diluted to provide a low density of reactivegroups that are typically present on a substrate surface at a density ofreactive groups of greater than 1/1×10⁶ nm² of surface area, but lessthan about 1/100 nm². In more preferred aspects, the density of reactivegroups on the surface will be greater than 1/100,000 nm², 1/50,000 nm²,1/20,000 nm² and 1/10,000 nm², and will be less than about 1/100 nm²,1/1000 nm², and 1/10,000 nm². For certain preferred applications, thedensity will often fall between about 1/2500 nm² and about 1/300 nm²,and in some cases up to about 1/150 nm².

The conditions for the attachment of the molecule of interest can becontrolled such that, for example, only one molecule of interest or oneactive molecule of interest is delivered to one or more opticalconfinements on a surface. In some cases, the conditions for theattachment of the molecule of interest are controlled such that 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the opticalconfinements have only one molecule of interest or one active moleculeof interest. Approaches for obtaining a high fraction of opticalconfinements having one molecule of interest is described in copendingU.S. patent application Ser. No. 12/384,097, which is incorporatedherein by reference for all purposes.

Phospholipid chemistries can also be used to functionalize the surfaceof the transparent or silica-based portions of the substrate.Chemistries using phospholipid compositions, have shown the ability, inthe presence and absence of calcium, to form different levels ofsupported phospholipid bilayers on metal oxide surfaces and silicondioxide based surfaces. By selecting the lipid composition and thepresence or absence of calcium, one can target deposition of molecules,either as blocking or coupling groups, onto the different surface types.For example, one can select a phospholipid that has high bindingselectivity for metal oxide surfaces and use it to block the metalportion of the surface. Alternatively, one can utilize a phospholipidwith an appropriate coupling group that has high binding selectivity forthe underlying glass substrate, and thus selectively couple additionalgroups to the surface of the transparent or silica-based portion of thesubstrate. Examples of these selective phospholipid compositions aredescribed in, e.g., Rossetti, et al., Langmuir. 2005; 21(14):6443-50,which is incorporated herein by reference in its entirety for allpurposes.

The molecules of interest are generally attached to the coupling agentsselectively placed onto the transparent or silica-based portions of thesurface as described above. A variety of chemistries are available forspecifically attaching a molecule of interest to the coupling agentsbound to the surface.

For example, where biotin is bound to the transparent or silica-basedregions of the surface, this surface can be used to attach the moleculeof interest using a binding agent such as streptavidin, which has a veryhigh affinity for biotin. In one approach, the molecule of interest hasa biotin tag which can then be attached to the surface using anintermediate binding agent, e.g., streptavidin, which acts to bind toboth the surface and the molecule of interest. In another approach,streptavidin is attached directly to the molecule of interest.

A variety of analytes can be delivered to reaction/observation regionsusing the methods and compositions herein. These include enzymesubstrates, nucleic acid templates, primers, etc., as well aspolypeptides such as enzymes (e.g., polymerases).

Similarly, a wide variety of proteins, e.g., enzymes, can also bedelivered using the methods herein. A variety of protein isolation anddetection methods are known and can be used to isolate enzymes such aspolymerases, e.g., from recombinant cultures of cells expressing therecombinant polymerases of the invention. A variety of protein isolationand detection methods are well known in the art, including, e.g., thoseset forth in R. Scopes, Protein Purification, Springer-Verlag, N.Y.(1982) and Handbook of Bioseparations, Academic Press (2000). Sambrook,Ausubel, Kaufman, and Rapley supply additional useful details.

For a description of polymerases and other enzymes that are active whenbound to surfaces, which is useful in single molecule sequencingreactions in which the enzyme is fixed to a surface (e.g., to a particleor to a wall of a reaction/observation region, e.g., in a ZMW), e.g.,conducted in a ZMW, see Hanzel et al. ACTIVE SURFACE COUPLEDPOLYMERASES, WO 2007/075987 and Hanzel et al. PROTEIN ENGINEERINGSTRATEGIES TO OPTIMIZE ACTIVITY OF SURFACE ATTACHED PROTEINS, WO2007/075873). For a description of polymerases that can incorporateappropriate labeled nucleotides, useful in the context of sequencing,see, e.g., Hanzel et al. POLYMERASES FOR NUCLEOTIDE ANALOGUEINCORPORATION, WO 2007/076057. For further descriptions of singlemolecule sequencing applications utilizing ZMWs, see Levene et al.(2003) “Zero Mode Waveguides for single Molecule Analysis at HighConcentrations,” Science 299:682-686; Eid et al. (2008) “Real-Time DNASequencing from Single Polymerase Molecules” Science DOI:10.1126/science.322.5905.1263b; Korlach et al. (2008) “Selectivealuminum passivation for targeted immobilization of single DNApolymerase molecules in zero-mode waveguide nanostructures” Proceedingsof the National Academy of Sciences U.S.A. 105(4): 1176-1181; Foquet etal. (2008) “Improved fabrication of zero-mode waveguides forsingle-molecule detection” Journal of Applied Physics 103, 034301;“Zero-Mode Waveguides for Single-Molecule Analysis at HighConcentrations” U.S. Pat. No. 7,033,764, U.S. Pat. No. 7,052,847, U.S.Pat. No. 7,056,661, and U.S. Pat. No. 7,056,676, the full disclosures ofwhich are incorporated herein by reference in their entirety for allpurposes. In some cases, the enzyme can be covalently attached to thesubstrate through functional groups on the enzyme such as amine,carboxylate, or thiol groups, for example with NHS or maleimide linkingchemistry.

In order to attach an enzyme to the surface, binding elements can beadded to the polymerase (recombinantly or, e.g., chemically) including,e.g. biotin, avidin, GST sequences, modified GST sequences, e.g., thatare less likely to form dimers, biotin ligase recognition (BiTag)sequences, S tags, SNAP-tags, enterokinase sites, thrombin sites,antibodies or antibody domains, antibody fragments, antigens, receptors,receptor domains, receptor fragments, ligands, dyes, acceptors,quenchers, or combinations thereof.

Multiple surface binding domains can be added to orient the polypeptiderelative to a surface and/or to increase binding of the polymerase tothe surface. By binding a surface at two or more sites, through two ormore separate tags, the polymerase is held in a relatively fixedorientation with respect to the surface. Further details on attachingtags is available in the art. See, e.g., U.S. Pat. Nos. 5,723,584 and5,874,239 for additional information on attaching biotinylation peptidesto recombinant proteins.

By controlling the starting dimensions of the chip and the oxidationconditions, the size of the island and the distance between the islandand the walls of the ZMW can be controlled. The process can be used toproduce an island of functionalizing agent which is small enough thatonly a small number of molecules of interest can be effectively bound.In some cases, the island of functionalizing agent can be small enoughthat only one molecule of interest can become effectively bound to eachZMW. For example, one can begin with a ZMW array on fused silica with analuminum layer of about 100 microns having apertures with circularcross-sections with diameters of about 60 nm. An oxidation at about 20 Vin a polyphosphonates electrolyte will result in a ZMW diameter of about82 nm, having an oxide layer with a hole with a diameter of about 5 nm.This substrate can then be treated with a functionalizing agent such asbiotin-PEG-silane. The oxide layer is subsequently removed using an acidbath. The result of this process is a ZMW having a diameter of about 82nm having an island of biotin functionality of about 5 nm. A molecule ofinterest, such as a polymerase enzyme-template-primer complex can thenbe bound to the island of functionality. Where the radius of gyration ofthe molecule of interest is greater than about 5 nm, the binding of onemolecule of interest will block subsequent binding, allowing for theproduction of an array of ZMWs, each having one molecule of interest atloadings greater than the 37% which can be obtained usingPoisson-limited analyte loading.

In some cases, it is desirable to have both the island of functionalityand a non-reflective layer on the walls of the ZMW. This can beaccomplished, for example by incomplete dissolution of the oxide layerto leave a portion of the oxide layer on the walls. Alternatively, thiscan be accomplished by subsequently adding a non-reflective layer by themethods described herein after the formation of the island anddissolution of the oxide layer. The non-reflective layer can be producedfor example, by performing a second oxidation after the formation of theisland of functional agent.

The binding of one or more molecules of interest to the island can becarried out either before or after step III. If the molecule of interestis inhibited or degraded by the conditions used to dissolve the claddingwalls, it will generally be attached after step III.

Step III can be carried out using compounds which selectively removeoxide from a metal surface. In some cases the oxide is removed with anacidic aqueous solution. Post-etch residue removers can be employed forthe selective removal of the oxide from the metal cladding. Residueremovers from Dupont, such as those from EKC Technologies, including theEKC600 series of materials can be used. In some cases, an aqueoussolution of EKC640 is applied at around 30 degrees C. to a substratewith an aluminum cladding to selectively remove aluminum oxide. In somecases compounds comprising phosphonic acids such as polyvinyl phosphonicacid or polyacrylic-polyvinylphosphonic acid are used to remove theoxide. Generally phosphonic acid compounds at a concentration from about0.1 percent to about 4 percent are used. In some cases, these compoundsare used at a concentration from about 0.1 to about 1 percent. In somecases, we have found that higher concentrations of phosphonic acid basedcompounds can provide superior results. Thus, in some cases, phosphonicacid compounds at a concentration from about 1.5 percent to about 3percent are used. At the higher concentrations, it is believed that theoxide becomes more unstable due, at least in part to the lower pH. Insome cases a pH of less than 4 is desired. Phosphonic acids are usefulas they can be formulated to have a high reactivity with metals such asaluminum, but very little reactivity with silica or functionalizedsilica surfaces, such as silane treated silica surfaces.

Arrays of Nanostructures

The methods described herein can also be used to produce an array ofnanostructures on a substrate. This can be accomplished, for example, byattaching a nanoparticle to a patch of functionalizing agent produced onthe surface. In one exemplary embodiment, in step II of FIG. 13,treatment with thiol-alkane-silane can be used to produce a patch ofthiol functionalizing agent. Treatment of the structure of FIG. 13(C)with gold nanoparticles will result in only particles that can fit intothe channel will become attached. In addition to only having one goldparticle attached per functional patch, the walls of the oxide canassist in localizing the particle. Alternately, the structure of FIG.13(D) can be treated with gold nanoparticles having a size on the orderof the functionalized island. This will result in the attachment of onlyone nanoparticle per island, and the attached particle will prevent theattachment of subsequent particles.

One aspect of the invention is a method for producing an array ofnanostructures comprising: providing a substrate having a top surface,the top surface having an aperture layer, the aperture layer having aplurality of apertures extending through the aperture layer to thesubstrate, the apertures having one or more cross-sectional dimension;oxidizing the substrate whereby an oxide layer is formed on the aperturelayer, whereby a cross sectional dimensions of the aperture is broughtto 50 nm or smaller; treating the substrate with a functionalizing agentwhereby the functionalizing agent becomes attached to the exposedportions of the substrate; exposing the substrate to nanostructures toattach the nanostructures to the functionalizing agent attached to thesubstrate; and dissolving the oxide layer.

Oxidation to Produce Nanopores of Controlled Size

In some cases, it is desirable to produce nanopores having controlledpore dimensions. Conventional methods of forming microstructures, suchas photolithography and etching may not be effective at formingnanopores having dimensions on the order of 10 nm. One aspect of theinvention is a method of forming an array of nanopores by usingoxidation to reduce the dimensions of larger holes.

One aspect of the invention is a method for forming an array ofnanopores comprising: providing a substrate comprising an array ofapertures extending therethrough, the apertures having one or morecross-sectional dimensions; and oxidizing the substrate whereby an oxidelayer is formed on the substrate whereby the formed oxide lowers anaperture dimension to 20 nm or less.

FIG. 15 illustrates an embodiment of the invention in which an array ofnanopores is formed by oxidizing a substrate having an array of holes toform an oxide layer such that an array having smaller nanoscale holes isformed. FIGS. 15(A) and 15(B) show a side view and a top viewrespectively of a portion of a substrate 1510 having an array holes orapertures 1520 extending therethrough. The substrate is oxidized, forexample by the methods described herein such as by thermal, plasma, orelectrochemical oxidation to produce a layer of oxide, resulting in thestructure shown in FIGS. 15(C) and 15(D). The resulting substrate has alayer of oxide 1530 that lowers the diameter of the hole or aperture1540. The resulting aperture has cross sectional dimensions on the scaleof nanometers. The starting hole or aperture 1520 can have, for example,a cross sectional dimension of about 500 nm to about 20 nm. Theresulting hole after oxidation 1540 can have, for example, a crosssectional dimension of about 100 nm to about 1 nm. The methods of theinvention are particularly useful for producing apertures of 20 nm orless, 10 nm or less, 5 nm or less, 2 nm or less, or 1 nm or less.

The cross-sectional dimension after oxidation can be, for example, 75%,50%, 40%, 30%, 25%, 10%, 5%, or 1% of the cross-sectional dimensionprior to oxidation. The final cross-sectional dimension can be fromabout 1% to about 75%, or about 5% to about 50% of the cross-sectionaldimension after oxidation.

The nanopores can have any suitable cross-sectional shape. Thecross-sectional shape can be, for example, circular, elliptical, square,or slits. In some cases, the apertures have a cross-sectional shape thatis a circle, and the cross-sectional dimension is the diameter of thecircle.

For example, one can begin with an array of holes in an aluminum layerof about 100 microns in thickness having apertures with circularcross-sections with diameters of about 60 nm. The aluminum layer mayhave a support layer that supports the aluminum layer structurally,while leaving the holes accessible. An oxidation at about 20 V in apolyphosphonates electrolyte will cause the buildup of oxide within theholes resulting in the production of a hole with a diameter of about 5nm.

The substrate in which the nanopores are formed can be any suitablematerial on which a stable oxide can be formed. The substrate can be,for example, a metal such as aluminum. The substrate can be asemiconductor such as silicon. The methods described herein forcontrolled oxidation of can be used to produce nanopores.

The nanopores of the invention can be used, for example, in sequencingapplications which involve passing a nucleic acid strand through thenanopore. The invention allows for the efficient production of arrays ofnanopores having controlled, small dimensions. The methods allow for thefine-tuning of the dimensions of the nanopore by controlling theoxidation conditions.

EXAMPLES Example 1 Electrochemical Anodization of ZMW Array

Electrochemical oxidation in the presence a poly(vinyl phosphonic acid)(PVPA) electrolyte was carried out using a three-electrodeelectrochemical cell equipped with a graphite counter electrode and asaturated calomel reference electrode. The metal cladding of the opticalconfinement array acted as the working electrode. FIG. 16 shows a cyclicpolarization curve for the anodization of an anodization carried outwith a solution of poly(vinyl phosphonic acid) (PVPA). The currentdensity stabilized at a value of 10 μA per cm², indicating the formationof a passive oxide layer. Upon returning the voltage to the open circuitpotential, the current drops, indicating that a passivation layer hasformed on the Al surface. Scanning electron microscope (SEM) analysis ofa cross-section of the ZMW after the electrochemical anodization shows athick layer of oxide. In some cases, a thin bright line is seen which isbelieved to be a phosphonate-rich passivation coating. The improvedcorrosion resistance of the ZMW can be seen using impedancespectroscopy. FIG. 17 shows electrochemical corrosion measurementscarried out in the buffers that are used for sequencing. Curve 1 is aZMW that was not passivated. Curve 2 is a ZMW passivated with PVPAthermally (without electrochemical treatment). The impedancespectroscopy results show that the electrochemical anodization in thepresence of phosphonates results in a significant improvement incorrosion resistance. Exposure of the electrochemically passivatedsurface to labeled neutravidin beads and to polymerase showed that thepassivated surfaces were also inert with respect to deposition ofbiomolecules such as proteins.

Example 2 Anodization of a Wafer

A fused silica wafer having an aluminum cladding layer with arrays ofapertures having diameters of about 100 nm formed on the wafer iscleaned by sonication in acetone, after which it is dried, and subjectedto a light plasma etch. Approximately 1 L of a 0.4% solution ofpoly(vinylphosphonic acid) is added to a 1 L beaker. The wafer isclipped to a wire connected to the anode of a power supply, and agraphite sheet is clipped to a wire connected to the cathode of thepower supply, and both the wafer and the graphite sheet are immersedinto the beaker. The power supply is turned on, and the desired voltageis applied. After 3 to 5 minutes, the power supply is turned off, andthe wafer is removed and rinsed with water and dried. The wafer can thenbe diced into chips sized about 9 mm by 9 mm for subsequent evaluation.

Example 3 TEM Images of ZMWs Having Non-Reflective Layers on their Walls

ZMW structures having non-reflective walls were produced byelectrochemical anodization as described herein. The resulting arrayswere prepared for transmission electron microscopy (TEM). Samples fortransmission electron microscopy were cross-sectioned using a dual beamfocused ion beam (FIB). Prior to sectioning, layers of platinum andchromium were deposited on the substrates.

FIG. 18 shows a TEM image of a cross section of a ZMW structure of theinvention prepared in the presence of PVPA by applying 10V. FIG. 18(A)shows that the ZMW has a layer of oxide on its walls and on the topsurface of the cladding. FIGS. 18 (B) and (C) show close up view of thestructure. The thickness of the oxide layer by TEM was determined to beabout 21 nm. The thickness of the oxide layer as determined byellipsometry was about 18 nm. FIG. 19 shows another SEM of the ZMWproduced by oxidation at 10 V in which the dimensions are shown. Thethickness of the cladding layer is about 93 nm, the thickness of thenon-reflective oxide layer is about 21 nm, the diameter of the ZMW isabout 132 nm, and the diameter of the region within the non-reflectivelayer is about 92 nm.

FIG. 20 shows a TEM image of cross-section of a ZMW structure producedby oxidation in the presence of PVPA at 15 V. The thickness of the oxidelayer is about 26 nm.

FIG. 21 shows a TEM image of cross-section of a ZMW structure producedby oxidation in the presence of PVPA at 25 V. The thickness of thecladding layer is about 79 nm, the diameter of the ZMW is about 195 nm,and the diameter of the region within the non-reflective layer (thesolution volume) is about 105 nm. The thickness of the oxide layer isbetween about 43 and 45 nm.

Example 4 Illumination Intensity Vs. Wall Material Refractive Index

A simulation was performed to calculate the illumination intensity inthe region of the ZMW inside the non-reflective layers (the solutionvolume). FIG. 22 shows a plot of illumination intensity for a greenlaser (diamonds) and a red laser (circles). As can be seen from theplot, for the red laser, the illumination intensity increases with wallrefractive index from about 1.3 to about 3. The illumination intensityincreases for the green laser from a wall refractive index of 1.3 toabout 2.5.

Example 5 Single Molecule Sequencing in ZMWs Having Non-ReflectiveLayers

A zero-mode waveguide array having about 3000 apertures through a 100 nmlayer of Al on fused silica was used to characterize the influence ofoxide thickness on DNA sequencing performance. Oxides of severalthicknesses were generated using the electrochemical methods describedabove. In the current example, 10V and 20V were applied to ZMW arrays togenerate 20 nm and 30 nm of aluminum oxide, respectively, on thealuminum substrate and inside the walls of the ZMWs. Following oxidegeneration, the substrates were treated with a specific biasing agent toprevent non-specific adsorption of sequencing components and tospecifically immobilize the sequencing polymerase on the fused silicabottom of the ZMW (see, e.g. U.S. patent application Ser. No.11/731,748, filed Mar. 29, 2007). Prior to sequencing, a single DNApolymerase molecule having a biotin label is complexed with a doublestranded and primed DNA to form a polymerase-template-primer complex.This complex is immobilized on functionalized fused silica substrates ofthe zero-mode waveguides using streptavidin or neutravidin. Thezero-mode waveguide array is exposed to a solution comprising labelednucleotide bases. Each base is labeled with a unique fluorescent marker(organic dye) that serve as signatures for detection, 555-T, 568-G,647-A and Cy5.5-C. Following immobilization of the DNApolymerase/template complex, the four bases in equal concentrations inbuffer are added to the system along with manganese to catalyze thereaction. The fluorophores are excited by 532 nm and 641 nm lasers.Fluorescence emission is monitored using a cooled CCD camera and thetime averaged spectra were converted to trace data acquired prior todata acquisition. FIG. 23 shows sequencing data in number of detectedphotons versus time, for a representative ZMWs for a control, and forarrays anodized at 10V and 20V. FIG. 24 shows signal to noise(Pkmid/Sigma) data for each of the four bases for a control, and forarrays anodized at 10V and 20V. FIG. 25 shows the distribution ofsignal-to-noise of sequencing pulses across a 3000 ZMW array for acontrol, and for arrays anodized at 10V and 20V. The data has beenanalyzed using base calling software in order to correlate the observedpeaks with the labeled bases that comprise DNA, (A) adenine, (C)cytosine, (G) guanine and (T) thymine. As can be seen in the figures,the signal-to-noise (defined as the fluorescence intensity correspondingto analog incorporation divided by the diffusion background) of the redfluorophores (647-A) are markedly enhanced with increasing oxidethickness. In this example, the SNR was increased over that of thecontrol by 1.5× and 2.25× for the 20 nm and 30 nm oxide thickness,respectively. The enhanced SNR enables improved sequencing accuracy bydecreasing the fraction of dark analogs that may otherwise fall belowthe detection threshold of the sequencing system.

Although described in some detail for purposes of illustration, it willbe readily appreciated that a number of variations known or appreciatedby those of skill in the art may be practiced within the scope ofpresent invention. To the extent not already expressly incorporatedherein, all published references and patent documents referred to inthis disclosure are incorporated herein by reference in their entiretyfor all purposes.

What is claimed is:
 1. A method for producing waveguides havingapertures that extend into a transparent substrate that are selectivelyfunctionalized on their bases comprising: providing an array of zeromode waveguides comprising an opaque layer on top of a silica-basedtransparent substrate layer wherein the zero mode waveguides comprisenanoscale apertures extending through the opaque layer and extendinginto the transparent layer, each zero mode waveguide having walls and abase; coating the array of zero mode waveguides such that the walls andthe bases are coated with a non-reflective layer; etching back thenon-reflective layer to expose the transparent substrate at the bases ofthe zero mode waveguides, thereby producing a non-reflective layerselectively on the walls of the zero mode waveguides and on the walls ofthe apertures extending into the transparent substrate layer, andtreating the array with an agent that selectively functionalizes theaperture bases comprising exposed silica-based transparent substratelayer to produce an array of zero mode waveguides having aperturesselectively functionalized on their bases.
 2. The method of claim 1wherein the coating process comprises a conformal coating.
 3. The methodof claim 1 wherein the etching back is performed using photolithographyto define the region for etch back.
 4. The method of claim 1 whereinbases of the apertures of the zero mode waveguides are selectivelyfunctionalized using a silane.
 5. The method of claim 1 wherein thecoating is performed using chemical vapor deposition (CVD).
 6. Themethod of claim 1 wherein the coating is performed using atomic layerdeposition (ALD).
 7. The method of claim 1 wherein the coating isperformed in the liquid phase.
 8. The method of claim 1 wherein thenon-reflective layer comprises an oxide.
 9. The method of claim 8wherein the oxide comprises Al₂0₃.
 10. The method of claim 1 wherein thenon-reflective layer comprises a polymer.
 11. The method of claim 10wherein the polymer comprises a polyimide.
 12. The method of claim 1wherein the array comprises more than 10,000 zero mode waveguides. 13.The method of claim 1 wherein the opaque layer comprises a metal. 14.The method of claim 1 wherein the zero mode waveguide has a diameterbetween about 1 nm and about 300 nm.
 15. The method of claim 1 whereinthe zero mode waveguide has a diameter between about 10 nm and about 200nm.